Forensically acceptable determinations of gestational fetal exposure to drugs and other chemical agents

ABSTRACT

Methods for collecting neonatal meconium samples, preparing meconium specimens for testing and for chemically analyzing neonatal meconium samples to determine their chemical composition are provided. A novel extraction method is employed in accordance with the invention to provide a non-aqueous, concentrated &#34;cocktail&#34; meconium extract containing substantially all of many possible target analytes in a single extraction step. Preliminary screening by fluorescence polarization immunoassay methods may be performed on the cocktail extract to qualitatively determine the presence of the target analytes in the meconium sample. If a positive preliminary result is obtained, new and improved quantitative GC/MS confirmatory procedures are provided by this invention to unequivocally identify and quantitate the amount of target analyte present in the sample in terms of nanograms of analyte per gram of meconium tested.

This application is a continuation of application Ser. No. 843,526,filed Feb. 28, 1992, now abandoned.

BACKGROUND OF THE INVENTION

The present invention generally relates to drug testing methods fordetermining maternal drug use during pregnancy and fetal exposure todrugs in utero. More particularly, it relates to new and improvedqualitative and quantitative methods for testing neonatal meconiumtissue samples to provide unequivocal evidence of prenatal exposure tochemical agents such as drugs of abuse by identifying and quantifyingthe presence of these chemical agents or their known metabolites in thenewborn meconium tissue samples.

There is growing concern in society today about the increasing numbersof newborn infants who are born in an undesirably unhealthy conditionbecause they have been exposed to harmful chemical agents in utero.Perhaps the most widespread concern surrounds an apparent increase inmaternal abuse of addictive drugs, both licit and illicit, duringpregnancy. Primary interest has been focused on the so-called drugs ofabuse, e.g., cocaine, opiates, including heroin, morphine and codeine,amphetamines, phencyclidine (PCP), and marijuana-related compounds, suchas tetrahydrocannabinoids (THCs). Secondarily, other so-called legallyavailable substances such as barbiturates, muscle relaxers, anti-anxietyagents, i.e. diazepam or lorazepam, alcohol, nicotine and other chemicalagents may be shown to affect the health of a developing fetus. Althoughthe present invention is primarily directed to drugs and their knownmetabolites, other chemical agents capable of becoming protein-bound mayalso be of interest. For example, gestational exposure to certaininsecticides, herbicides, industrial chemicals, air pollutants and waterpollutants may also be shown to adversely affect the exposed fetus inutero and the health of the newborn infant.

Maternal abuse, use or exposure to several known chemical agents, suchas addictive drugs has been reported to lead to decreased birthsize andbirthweight, decreased head circumference, decreased gestationalduration and in a few cases, congenital defects. Generally, infants bornto drug addicted and/or using mothers may be characterized by beingcompromised, in ill-health, exhibiting an early failure to thrive andpossibly may be expected to show significant developmental delays oranomalies if they survive after the first few days and weeks. Forexample, neuronal damage may not become recognizable until later in thelife of the child.

Proper medical care and treatment for the special needs of thesedisadvantaged individuals frequently requires that treating physiciansand nurses be advised of the fact that the neonate has been exposed inutero. Moreover, the identity of which drug or chemical agent the childhas been exposed to as a fetus may also be critical to early courses oftreatment and ultimate survival. If latent neuronal damage was causedleading to real or apparent learning disabilities, early detection andpossession of the information can really help that newborn in many ways.If learning disability problems are inevitable or likely, early extraeducational efforts to assist the child may help to reduce or eliminateany adverse effects to that child. Moreover, if the child is identifiedearly as disabled or handicapped due to in utero drug exposure, Federaleducational and social assistance may be available to the child to helpwith the costs of raising, educating and caring for the child. Thefinancial and human costs of delivering the high-risk neonatal medicalcare and continuing medical and social care and services for theseinfants in increasingly higher numbers is staggering.

Recent studies show that maternal abuse of addictive drugs duringpregnancy is widespread and increasing. The National Association forPerinatal Addiction Research and Education estimated that approximately11% of women studied in 36 hospitals had used illicit drugs duringpregnancy. In regional areas where drug abuse is known to be asignificant problem, the estimates are considerably higher. Recently, inone southern California hospital, authorities estimated that they haddelivered at least about 20,000 drug addicted babies in one year.Similarly, a Chicago Municipal hospital estimated delivery of over12,000 drug-exposed or addicted newborns per year. Conservativeestimates by public health experts indicate that there are at leastabout 500,000 drug-exposed neonates born each year in the United States,about 10% of the annual birth rate.

The severity of this public health and societal problem has come to theattention of several legislatures. Some jurisdictions proscribe the useof addictive drugs during pregnancy and define the presence of suchdrugs in the neonate as a prima facie child abuse, thereby creating animmediate state interest in the care and custody of these infants- Inaddition to increased demands for intensive medical care and management,health care providers must also act responsibly in accordance with newlydefined and increased duties imposed on them by these new statutes andregulations. Health care providers in some jurisdictions have a duty toscreen and identify the drug exposed neonates and their mothers.

Earlier efforts to identify and assess those mothers who used or abuseddrugs during pregnancy and who thereby exposed their infant newborns todrugs in utero have included obtaining oral admissions from the mother,blood testing and urine testing. Oral histories, as has been mentionedabove, are generally not forthcoming or reliable. Testing of bloodtissue samples for the presence of drugs or their metabolites isgenerally time consuming and expensive. The recent AIDS epidemic hasmade collection, testing and handling of blood tissue samples unwise andundesirable. In addition, the target analytes of the drugs of abuse,typically looked for will not be present in either maternal bloodstreamor the neonate's bloodstream even only six hours after the mother hastaken the drug. Usually the first neonatal blood samples aren't takenuntil about 12-24 hours after delivery. Accordingly, blood testing of aninfant newborn is expected to provide a ridiculously large number offalse negative results.

Urine testing is also problematic for several reasons. Collectingadequate sample specimens from premature infants is difficult at best.Unless the fetus was exposed to the drug within one to three daysimmediately prior to delivery which is generally unlikely, urine screensfrequently yield false negative results. Accordingly, urinalysis isgenerally inconclusive at best.

More recently, it has been demonstrated that meconium tissue formed inthe intestines of a fetus in utero may provide the best physicalevidence of maternal drug use. Meconium is a complicated heterogeneoustissue including a matrix of proteinaceous and cellular solids capableof binding, adsorbing and storing various chemical agents per se and/ortheir respective metabolic end products. Meconium is continuously formedand stored in the fetal intestines from late in the first trimester ofgestation i.e., from between about 12-16 weeks, until birth.

Meconium forms the first several excreta of a newborn infant until achange to transitional milk stools is observed. Meconium tissue maytherefore act as a biological time-capsule, in that each infant'smeconium may reveal the history of the fetus in utero in terms of itsexposure to various chemicals which become bound in the meconium matrixas the meconium is formed. The contents of neonatal meconium may provideevidence of exposure to drugs or other chemical agents back in timeduring the pregnancy, possibly as early as the later part of the firsttrimester and definitely from the second and third trimestersimmediately before birth. The importance of the meconium tissue is thatthis precious sample is a one-shot deal. Unless the meconium is properlycollected and analyzed a great deal of once-in-a-lifetime information ispermanently lost. The personal and social implications of not having theimportant information the tissue can provide are very serious.

A major problem associated with collecting and studying meconium tissueis that it is extremely difficult to work with. Meconium has a thickvisco-elastic nature that does not readily lend itself to typical sampleanalysis methods and equipment.

In U.S. Pat. No. 5,015,589 to Ostrea Jr., two distinct methods fortesting a meconium sample to qualitatively determine whether or not itcontains morphine and/or cocaine metabolites or cannabinoids,respectively, are described. In the assay procedure to qualitativelyscreen infant meconium for the presence of morphine or cocainemetabolites, a 0.5 to 1.0 gram sample of infant meconium is diluted morethan 2 to 1 with an aqueous hydrochloric acid solution. The acidifiedand diluted sample is vortexed and poured through a glass wool filter toremove gross particulates. The filtrate is centrifuged and an aliquotportion is analyzed for morphine metabolites and cocaine metabolites byradioimmunoassay.

In the Ostrea Jr. method for qualitatively determining the presence ofmarijuana metabolites, a neonatal meconium sample is admixed withabsolute methanol, vortexed and permitted to stand at room temperaturefor several minutes. Thereafter, the mixture is filtered, centrifugedand an aliquot portion of the supernate is tested for cannabinoidmetabolites by radioimmunoassay. The Ostrea Jr. methods described inU.S. Pat. No. 5,015,589 suffer from a number of shortcomings. Thedeterminations are qualitative at best and do not offer unequivocalquantitative evidence of prenatal drug exposure needed to meet currentforensic standards. Moreover, the qualitative procedures described aregenerally not reproducible in practice. This is believed to be primarilycaused by the broad compositional variations in the heterogeneousmeconium tissue in general. For example, a positive or negative resultmay depend on the location from which the aliquot portion of themeconium sample was taken in relation to the overall meconium specimen.This distributional variation within a single sample can often lead tofalse negative test results. Furthermore, the procedures are notparticularly sensitive in practice. In accordance with the Ostrea Jr.method, a small sample of meconium is diluted with an aqueous acid.Ostrea Jr.'s acid reagents dilute trace amounts of target analytes foundin the sample to a large degree. After the immunoassay reagents areadded, the target unknowns are diluted even further, often below theminimum detectable concentrations of the immunoassay reagentsthemselves. The aqueous mineral acids employed are so strongly acid thatthey not only hydrolyze and denature the meconium matrix to releasetarget analytes into aqueous solution, these acids may also de-naturethe recognizable structure of the metabolites being assayed, furtherlowering their effective detectable concentrations.

Poor sensitivity and non-reproducibility experienced with the Ostrea Jr.method may also result from the fact that Ostrea Jr. requires a glasswool filtration step to remove gross particulates from the acidifiedmixture. It has independently been observed that untreated glasssurfaces, such as those presented by the glass wool filters employed inthe Ostrea Jr. method, are reactive with the sample analytes and absorband bind target substances, thereby taking them out of the assaysolutions. Untreated glass surfaces can absorb as much as 10 nanogramsof drug or other metabolite for every square centimeter of untreatedglass surface area that the drug containing solution is exposed to.

Another disadvantage to Ostrea Jr.'s methods is that differentextraction procedures and steps must be performed on the same meconiumtissue for each target analyte in order to only make what is anessentially unreliable qualitative determination for that analyte. In acommercial setting, too many separate procedures and duplicative stepsdisadvantageously increase the time and costs needed to implement theOstrea Jr. method. Finally, the Ostrea Jr. patent describes making theirscreening determinations using radioimmunoassays which are undesirablebecause costly special care, handling and disposal steps are required inorder to use these radioactive materials.

Another prior art method for screening neonatal meconium samples isdescribed by Rosenzweig et al. in an abstract appearing in ClinicalChemistry, Vol. 36, No. 6, (1990) at page 1023, Abstract No. 0334. Inthe method described in this abstract, evidence of drugs and drugmetabolites may be qualitatively determined for amphetamine, cocainemetabolite, morphine metabolite and cannabinoid metabolite in neonatalmeconium samples by non-radioactive immunoassay methods. In accordancewith this screening method, a meconium sample is extracted withmethanol, centrifuged and the supernatant is filtered through filterpaper. The volume of the filtrate is reduced by evaporation andreconstituted in a phosphate buffer. The buffered sample is thenanalyzed by enzymatic colorimetric immunoassay or by a fluorescencepolarization immunoassay method.

The non-radioactive screening methods described by Rosenzweig also havedisadvantages in terms of reliability and sensitivity. The meconiummethanol extract is generally too highly colored for direct reading in acolorimetric read assay. Ten fold dilutions are needed to achieveworkable readability which reduces analyte concentration to at or belowsensitivity limits to the assays. The method includes a paper-filterfiltration step which has also been shown to act as a drug spongeundesirably absorbing target analyte out of the sample. Also, theRosenzweig methods described in the abstract are only qualitativescreens and do not provide the unequivocal quantitative evidence needed.Furthermore no special sample preparation steps are described orsuggested so that the disclosed method also suffers from lot to lotvariability for samples taken from the same patient sample.

In another abstract published in Clinical Chemistry, Vol. 36, No. 6(1990) at page 1022, Abstract No. 0327, Clark et al, describe a firstquantitative procedure for determining quantities of cocaine and itsmajor metabolite, benzoylecgonine, from meconium samples. The Clark etal. method includes extracting cocaine and its metabolites from a sampleof meconium with methanol and centrifuging to remove solids. Thesupernatant is concentrated by evaporation, reconstituted with phosphatebuffer and extracted with a solid phase cation exchange column. Solidphase bound analytes are eluted into an organic solvent, derivatized andinjected into a GC/MS instrument in selective ion monitoring mode.Unknown patient samples were compared with spiked negative samples andquantitative results were obtained from the standard curve. The GC/MSmethod was reported by Clark et al. to be able to detect cocaine or itsmetabolite at levels as low as 300 ng of drug per gram of meconiumtested.

The earlier GC/MS confirmatory method described by Clark et al. madesome progress toward providing a quantitative meconium testing methodbut suffers from several drawbacks. In practice, a methanol extract asdescribed by Clark et al. results in a meconium sample fluid containingneutral fats and free fatty acids which frequently clog the solid phaseextraction columns. The Clark et al. procedures also do not addressvariability of the meconium samples per se and do not provide forvariable dispersability of the meconium in the methanol so thatnon-reproducible results are often obtained. The sensitivity for theseconfirmatory procedures was stated to be a drug concentration of about300 ng/g of meconium for cocaine and benzoylecgonine. Nevertheless, theability to detect even smaller amounts of drugs or their metabolites anddetermining exposure as early as within the second and first trimestersof pregnancy is still desired. The Clark et al. article specificallydescribes a quantitative GC/MS method for cocaine only with limitedsensitivity. No teachings or suggestions are provided for makingquantitative GC/MS assays for meconium samples for other drugs of abuse,their respective metabolites or other chemical agents of interest.

Accordingly, to overcome the limitations of the prior art procedures andmethods, it is an object of the present invention to provide new andimproved testing methods for obtaining unequivocal evidence of prenatalexposure to selected target analytes, such as the drugs of abuse andtheir metabolites, as well as other chemical agents.

It is another object of the present invention to provide faster, lessexpensive and more reliable methods for testing neonatal meconiumsamples to qualitatively and quantitatively determine gestational fetalexposure to not only cocaine and its metabolites, but also to opiates,amphetamines, phencyclidines and cannabinoids, as well as other licitand illicit drug analytes.

It is still another object of the present invention to provide new andimproved methods for collecting and preparing neonatal meconium tissuesamples for further testing to provide improved analyte separation andrecovery.

It is still another object of the present invention to provide a new andimproved method for making a smooth, substantially uniform time-averagedmeconium tissue product for further testing to reduce the incidence offalse negative results and aliquot to aliquot variability for neonatalmeconium tissue specimens.

It is another object of the present invention to provide a new andimproved method for liberating and extracting substantially all targetanalyte values of interest from a neonatal meconium sample, inrelatively concentrated form in a single step, for use in a number ofanalytical testing procedures.

It is a further object of the present invention to provide new andimproved methods meeting currently accepted forensic standards tounequivocally identify and quantitate the presence of drugs of abuse inmeconium adapted for use on a commercial scale in a commercial testinglaboratory.

It is still another object of the present invention to improve thequality of medical care for newborn infants in our society by providingfast, inexpensive and reliable methods for early detection of thosenewborns exposed to various harmful chemical agents while a fetus inutero.

SUMMARY OF THE INVENTION

In accordance with these and other objects, the present inventionbroadly comprises new and improved methods for collecting meconiumsamples, preparing meconium specimens appropriate for testing, andchemically analyzing neonatal meconium samples to determine theirchemical composition. In accordance with the first aspect of theinvention, new and improved methods are provided for collecting themeconium tissue of a subject neonate in a manner which effectivelypreserves the chemical information stored in the meconium tissue and topromote maximum recovery of sample target analyte.

More particularly, in accordance with this aspect of the invention, aplurality of meconium excreta or bowel movements are collected from asubject newborn infant in diapers specially lined with a removable linermaterial selected for its non-absorbent and non-adherent properties withrespect to the meconium tissue. Each incremental meconium sample istransferred from the lined diaper to a storage container and kept underrefrigeration until the last meconium excreta movement has been made bythe infant. Meconium has a very characteristic bluish-gray-green colorand a rubbery elastic texture which differs greatly from the firsttransitional milk stool made after the infant begins postpartum feeding.The first transitional milk stool is characterized by being looselyformed and bright yellow tan in color. Accordingly, collecting meconiumuntil the first transitional milk stool is observed is an easy end pointto identify. The production of meconium excreta by infants varies frominfant to infant but can occur from birth up to as many as 10 days afterbirth. Collecting all of the meconium samples from a single baby andstoring them in a single refrigerated container provides an extended"time window" tissue sample which may be analyzed to provide evidence ofdrug exposure.

In accordance with the preferred embodiment, the collection andpreparation procedure further includes the step of smoothing out themeconium samples to allow target analytes such as drugs which are notuniformly distributed through the meconium to be more thoroughly andevenly distributed in a prepared sample. Redistributing drug analytes toform a time-averaged tissue sample is achieved by rendering the meconiuminto a smooth substantially uniform non-striated paste-like mass.Pre-processing pooled meconium specimens enhances reproducibility oftesting results for the tissue and avoids the occurrence of falsenegatives brought about by distributional anomalies inherent in thenature of meconium tissue itself.

In accordance with another aspect, the present invention provides a newand improved method for preparing a concentrated neonatal meconiumextract. The concentrated meconium extract is thereafter employed as thesample fluid for performing qualitative screen testing to determinewhether one or more target analytes are present in the original meconiumsample. Thereafter, in accordance with the overall method of the presentinvention, if a positive result from the preliminary screen testing isobtained, a quantitative test procedure is undertaken to definitivelyidentify and quantify target analytes present in the meconium sample inquantitative terms, i.e., terms of nanograms of analyte per gram ofmeconium.

More particularly, the new and improved method for preparing aconcentrated neonatal meconium extract comprises providing a sample ofnewborn meconium suspected of containing at least one target analyte. Aminor effective amount of a substantially non-aqueous volatile organicacid is added to the prepared test sample to form a first mixture. Thefirst mixture is agitated for a time sufficient to release substantiallyall of the at least one target analyte from the meconium samplesubstantially without diluting the sample with water.

In accordance with an especially preferred method, after the volatileorganic acid is added to a pre-processed meconium specimen sample, thepooled and collected meconium tissue is next finely subdivided anddisrupted by high shear mixing, preferably by means of a shearing bladedinstrument, such as a tissue homogenizer, to disrupt the cellular andother protein structures of the meconium matrix. Shearing of theacidified pre-processed sample renders it more amenable to subsequentprocessing steps. Tissue homogenizing improves liberation of the targetanalytes and more thoroughly distributes the unbound target analytesevenly and uniformly within the overall tissue specimen to avoid sampleto sample variations in subsequent testing. Preferably, afterhomogenizing, the meconium test sample is a substantially uniformmeconium tissue sample homogenate having a finely divided, averageparticle size of less than about 10 microns, and especially preferablybetween about 2.0 to about 5.0 microns to facilitate release andextraction of the target analytes.

The volatile organic acid agent for use herein is generally acidicenough to begin to denature the meconium matrix to release the targetanalytes in liberated or unbound form. The agitation of the firstmixture should be fairly vigorous and result in a substantially uniformfirst liquid homogenate. After the first liquid homogenate is obtained,a minor effective amount of a volatile organic solvent is added to thefirst homogenate to form a two-phase mixture including an organic phaseand a second phase. The two-phase mixture is agitated and thoroughlymixed for a time sufficient to permit substantially all of the releasedtarget analyte to be extracted and collected into the organic phase.Thereafter the organic phase is separated from the second phase toprovide the concentrated extract. The volatile organic extract mayreadily be concentrated further by evaporating the volatile organiccomponents in a sample concentrator.

In accordance with an important aspect of this method the concentratedmeconium extract comprises a "cocktail" extract which containssubstantially all of the various target analytes being looked for. Forexample, if drug abuse testing is the object of the procedure, thevolatile organic acid/volatile organic solvent treatment step results ina concentrated non-aqueous extract containing substantially all of thetarget drug and drug metabolite analytes in a single extraction step.For example, target analytes of cocaine or its benzoylecgoninemetabolite, opiates including morphine and codeine, amphetamines,marijuana metabolites and phencyclidine all comprise protein-boundtarget analytes which are successfully collectively extracted into the"cocktail" extract in accordance with the new and improved method withadvantageously good recovery.

An important feature of this aspect of the invention is that the targetanalyte values extracted from the meconium matrix are not unnecessarilydiluted with water but are extracted in relatively concentrated form.The target unknowns may be readily concentrated even further uponstanding or by evaporating in a sample evaporator in a short period oftime of less than three hours. The volatile extract provides animportant advantage when scaling up drug testing in a commercial settingfor a commercial laboratory because prolonged aqueous dilution andprolonged or high-temperature drying steps are avoided.

After the concentrated "cocktail" extract is obtained, in accordancewith the method of this invention, aliquot portions of the extract arepreliminarily screened or tested in an homogeneous immunoassay method toqualitatively determine whether or not the extract contains the targetanalytes of interest. In accordance with the preferred embodiment of theinvention, the target analytes will be the drugs of abuse and theirrespective metabolites selected from cocaine, amphetamines, opiates,phencyclidine and cannabinoids and/or their respective metabolites. Thepreferred immunoassay technique is by fluorescence polarizationimmunoassay (FPIA). If a positive result is obtained from thepreliminary assay screen, then another aliquot portion of the preparedmeconium tissue sample is subjected to a further independentquantitative confirmation procedure which includes an extractionprocedure specific for that analyte. The confirmatory aliquot of sampleis spiked with labelled analyte analogs which are extracted selectivelywith their respective target analytes. The extracted analytes andanalogs are derivatized and subjected to quantitative analysis using gaschromatographic mass spectrometric (GC/MS) equipment.

In accordance with this confirmatory aspect, the present inventionprovides new and improved confirmatory (GC/MS) quantitative assayprocedures for not only cocaine and its metabolite but other drugs ofabuse such as the opiates, amphetamines, cannabinoids and phencyclidineas well. The earlier "cocktail" extract prepared in the preliminaryqualitative screening represents an effective compromise at getting allof the target analytes out of an aliquot portion of the sample in asingle extraction step to provide an easy one-step approach. However,the preliminary extraction reagents and methods may not necessarilyprovide the highest rate of extraction for each given target analyte inthe drugs of abuse group. Accordingly, for the confirmatory procedures,a second aliquot portion of the prepared meconium tissue sample is takenand a second extraction procedure is run which is designed to bespecific for the target analyte to now be quantitatively determined.

More particularly, the particular extraction procedure for the GC/MSconfirmatory extraction step is chosen to maximize the extractionrecovery of that particular target analyte already tested positively tobe present in the qualitative preliminary screen. The exact confirmatoryextraction procedures for each target analyte will be described ingreater detail hereinafter. Generally, the opiates and amphetamines areextracted using a liquid/liquid extraction. The improved cocainequantitative analysis procedure and the cannabinoid and phencyclidineconfirmatory procedures each employ solid phase extraction columns toprovide an appropriate extract fluid for GC/MS quantitative analysis.

The new and improved confirmatory extraction procedures and GC/MSquantitative analysis methods of the present invention now provideunequivocal evidence of prenatal drug exposure in a forensicallyacceptable manner to confirm the presence of cocaine metabolites, THCmetabolites, opiates, amphetamines and PCP in the neonate which wereheretofore unavailable for meconium testing. The methods of theinvention may also be used to quantify and provide unequivocal forensicevidence of exposure to other chemical agents. The methods of theinvention provide cheaper, faster and more sensitive assays which may bereadily adapted to a commercial laboratory setting. The new and improvedpreliminary cocktail screening extract procedure results in a ten-foldimprovement in sensitivity over prior art aqueous acid extraction andmethanol extraction methods.

Other objects and advantages of the present invention will becomeapparent from the following Detailed Description and illustrativeworking Examples.

DETAILED DESCRIPTION OF THE INVENTION

In greater detail now, the first aspects of the new and improved methodsof this invention relate to the proper collection and preparation of theneonatal meconium tissue samples preparatory to further testing. As hasbeen mentioned above, the advantage of testing meconium for drugs ofabuse or other target analytes lies in the fact that meconium isgradually formed and thereafter is stored in the intestines of theinfant until gradually expelled in the immediate postpartum time period.Overall, the entire meconium content of the infant newborn providesstored chemical information developed over most of the gestationalperiod of development for the infant. Accurate and sensitive chemicalanalysis for small amounts of specific target analytes present in themeconium samples requires the striated, non-uniform collected meconiumspecimens to first be vigorously mashed and stirred, to re-distributethe contents of the pooled meconium matrix so that a substantiallysmooth, evenly distributed putty-like pre-processed meconium product isformed. During collection, pooling and processing, care should be takento preserve the maximum amounts of target analytes and not to losesample analyte to various surfaces the samples may come in contact withat these stages of preparation.

In accordance with the preferred embodiment, a complete meconium sampleis collected from a subject newborn infant suspected of gestationalexposure to one or more target substances by collecting and pooling eachmeconium stool produced by that infant from birth until the firstappearance of transitional or milk stool appears. The meconium tissue iscollected in the babies' diapers and because the meconium tissue and anycontained target analyte may become absorbed on or into the diapersurfaces, in accordance with a preferred embodiment herein, each of thebabies' diapers is lined with a liner sheet of water-proof,non-absorbent material. The liner sheet may generally comprise any sheetmaterial having surface properties which effectively resist or avoidadsorption, absorption or reaction with the meconium sample. Plasticmaterials are preferred for forming the diaper liners and polyethylenesheets are especially preferred. Generally, in addition to not absorbingsample, the diaper liner sheet should be able to maintain its structuralintegrity in the wet diaper environment.

Each meconium bowel movement for the subject infant is collected on thediaper liner and thereafter stored in a lidded storage container havinga volume capable of receiving all meconium samples produced by thesubject neonate, typically from about 2.5 to about 5.0 grams. Meconiumgenerally has an apparent density such that about one teaspoon ofmeconium approximately weighs about one gram. A minimum of at leastabout 2.0 grams of meconium overall should be collected from eachneonate.

The collected meconium specimens should be transferred from the diaperliner to a non-surface reactive lidded storage jar or vessel and keptunder refrigerated conditions until sample collection for that infant iscomplete. In accordance with the preferred embodiment, the storagevessel is selected to be made from polystyrene. The storage containergenerally should not be made of untreated glass or other sample-reactivematerial to prevent sample and target analyte from becoming absorbed oradsorbed by the surfaces of the glass container. Collected and pooledspecimens stored in the container may be stored under refrigeration attemperatures of about 2° to about 8° C. for up to 30 days without anysignificant analyte loss. For longer storage times, pooled specimensshould be stored frozen at -15° C. or lower.

After all of the subject infant's meconium has been collected, then inaccordance with the preferred embodiments, the pooled meconium tissue atroom temperature is pre-processed to provide a time-averaged tissuesample to correct for the non-homogeneous nature of drug distribution inthe meconium mass. Vigorous stirring and mixing of the sample isperformed to improve the distribution of the target analyte in themeconium matrix. In practice, it has been found that the meconium matrixis tenaciously rubbery, striated and non-uniform and mixing should bethorough enough to be effective to achieve a substantially smooth,evenly distributed non-striated sample, characterized by low sample tosample variation.

In accordance with the method of the present invention, the smoothpre-processed meconium product is thereafter subjected to a two-stepanalytical approach and procedure to unequivocally identify and quantifythe presence of one or more target analytes in the meconium of thesubject infant. A first preliminary qualitative immunoassay screeningprocedure is performed to identify the presence of target analyte(s) inthe patient sample. For those infants testing positive for targetanalyte(s) in the preliminary screening, a second quantitativedetermination by a GC/MS procedure is run to quantify the amount of eachspecific target analyte present in the sample. The results of the GC/MSconfirmatory procedures report the drug or other target analyte findingsin acceptable quantitative units such as nanograms of target analyte pergram of meconium tested.

In accordance with a special feature of the new and improved method ofthe present invention, the preferred pre-processed meconium tissueproduct is subjected to a new and improved extraction procedure toliberate substantially all of the target analytes from the meconiummatrix and form a concentrated non-aqueous meconium "cocktail" extract.

More particularly, in accordance with the preliminary screeningprocedure, an aliquot portion of the pre-processed meconium sample issubjected to a generalized extraction treatment procedure to liberateand separate the target analytes from the meconium matrix inconcentrated form for further testing. The extraction procedure shouldbe fast, accurate and efficient and well suited to commercial laboratorysettings where large numbers of samples must be tested at highthrough-put rates. The extraction should liberate substantially alltarget analytes of interest in a single efficient step.

In accordance with the preferred embodiment of the present invention, anew and improved group extraction step is performed by placing about a 1gram sample of the pre-processed meconium product into a polypropylenetube (16 by 125). A minor effective amount of a substantiallynon-aqueous, volatile organic acid reagent is added to the pre-processedsample in the tube to form a first mixture. The volatile organic acid isselected to be effective to precipitate proteins and neutralize lipidsand fatty acids to aid in separation of the analyte values from themeconium matrix.

The meconium sample in accordance with the preferred embodiment, shouldbe subjected to high shear, bladed processing, to break up the meconiummatrix into cellular and sub-cellular sized fragments in the acidtreatment step to insure substantially complete liberation of all targetanalyte(s). The high shear processing treatment preferably results in asmooth, substantially uniform finely-divided soft paste-like producthaving an average particle size of less than about 10 microns.Especially preferably the pre-processed meconium tissue product will beprocessed until a substantially smooth, non-striated homogeneousmeconium product having an average particle size of between about 2.0 toabout 5.0 microns is obtained.

In accordance with this preferred aspect of the invention,pre-processing of the pooled meconium samples to finely-divided, evenlydistributed homogeneous form may be accomplished using a commerciallyavailable tissue homogenizer. A tissue homogenizer is an apparatusincluding a hollow cylindrical shaft having a small diameter so that itmay be fully inserted into a narrow tube such as a test tube or samplecollection vial. A rotatable blade mounted on a concentric axial shaftwithin the cylindrical outer tube, shears the tissue against the blades,itself and the interior surface of the tissue homogenizer tube to formthe disrupted, finely-divided processed meconium product. The tissuehomogenizer is moved through the meconium sample until processing andhomogenization are substantially complete, typically in a matter ofabout 5 minutes or less. A preferred commercially available tissuehomogenizer for use herein is an OMNI 5000 model tissue homogenizeravailable from OMNI International, Inc. In practice, it has beendiscovered that acid addition followed by mixing, even vortex mixing, isnot as effective at obtaining liberation of target analytes from themeconium matrix. Failure to homogenize or process in a fractionating,disruptive partitioning manner, has been observed to result insubstantially lower recoveries of target analytes.

It has also now been discovered that often significant quantities ofanalyte remain in the fat or lipid segments of the meconium. In the pastlipids have not advantageously been extracted because of the extractreagents used or because of the handling methods employed. In accordancewith this invention, the volatile organic acid should extract the lipidsand neutral fats in the first instance together with the target analytesto liberate substantially all target analyte values from the meconiummatrix. The acid should exhibit good volatility, i.e. high evaporationrates at or above room temperatures. The acid liberating agent shouldnot react with the analyte per se. Moreover, the volatile organic acidshould not liberate harmful or toxic fumes on evaporation orvolatilization.

In testing a number of non-aqueous organic acid candidates, the bestacids were found to be propionic and glacial acetic acids. Propionicacid was slower to evaporate and accordingly the especially preferrednon-aqueous volatile organic acid for use in the method of the presentinvention is glacial acetic acid. The preferred glacial acetic acidcomponent should be added in a minor effective amount of about 2 to 5ml. of concentrated glacial acetic acid per gram of sample andpreferably about 3 mls. of acetic acid are added.

Thereafter, in accordance with the preferred method of this invention, aminor effective amount of a volatile organic solvent is added to thefirst liquid homogenate to form a two-phase mixture which includes anorganic phase and a second phase. The volatile organic solvent should beeffective to dissolve and collect the liberated analyte values in themeconium sample and preferably will solubilize or dissolve fats orlipids to insure that all of the target analyte is carried over into theorganic phase. The two-phase mixture thus formed is thoroughly mixed fora time sufficient to permit all of the released target analyte values tobe extracted and collected into the organic phase. The volatile organicsolvent selected should be fast and cheap and volatilize readily toprovide a concentrated analyte extract. The organic solvent should alsoselectively extract all the target analyte and some of the free fats topromote maximum recovery of the target analyte from the meconium matrix.The organic solvent should be polar enough to extract metabolites ofmorphine or cannabinoids such as the glucurinide forms. In accordancewith the present invention, the preferred volatile organic solvent interms of volatility and cost and extractive properties is acetone,although acetonitrile or certain dialkylethers may also be used.

In accordance with this intermediate step, the two-phase mixture isthoroughly mixed by vortexing and thereafter is mixed on a sample mixerfor a period from about 3 to 10 minutes, preferably for about 5 minutes.Generally, the two-phase mixture should be agitated sufficiently topermit all of the target analyte(s) to be extracted and collected intothe organic phase.

In accordance with an especially preferred embodiment, the volatileorganic solvent reagent will also include a minor effective amount of aglass anti-binding agent in the form of a secondary or tertiary aminehaving a molecular weight of from about 100 to about 400 which isnon-reactive with the target analyte or other sample components. Thepreferred glass anti-binding agent for use herein comprisesdiphenylamine. In addition, all glassware to which the sample orextraction reagents come in contact should be silanized before samplematerials are placed therein. A more complete description of silanizingtreatment is set forth hereinafter.

After the two-phase mixture is thoroughly mixed by vortexing orotherwise for a period of about 5 minutes, the organic phase isseparated from the second phase by any suitable separation method. Thepreferred method for separating the organic phase from the second phasecontaining non-organic meconium solids and other materials is bycentrifugation. In accordance with this invention, the separation isperformed by centrifuging at high speed sufficient to provide greaterthan or equal to about 2,500 g of separation force for a period of fromabout 5 to 15 minutes, preferably 10 minutes until separation issubstantially complete. After centrifugation, the top organic acetonephase is transferred into a silanized concentration cup, leaving behindthe second phase containing the meconium solids. In accordance with aspecially preferred method, the supernatant fluid portion of thecentrifuged sample is carefully decanted through a polypropylene fritinto the concentration cup to insure removal of solids and largeparticulates. A polypropylene frit is used because it does not absorbsample and is substantially non-reactive with the sample and sampleanalyte.

Before evaporating the concentrated extract to further concentrate themeconium group or cocktail extract, a small amount of a sulfating agentis preferably added to convert certain volatile amphetamine analytes toa non-volatile sulfate form to thereby prevent or avoid their becomingentrained with the evaporating volatile solvents. A preferred sulfatingagent is a 0.1% sulfuric acid solution in methanol.

In accordance with this aspect of the present method, the volume of theextract is further concentrated from about 6 to 10 ml. down to 0.3 to0.6 ml. by heating the volatile extract in a sample concentrator atelevated temperatures at or above room temperatures and at pressures ator below atmospheric pressure. Preferably, the sample is concentrated at75° C. over air and vacuum. The sample concentrator may be commerciallyobtained from Alltech Corporation, Deerfield, Ill. and an Alltech Model190-A sample concentrator is especially preferred. A major advantage ofthe substantially non-aqueous, non-diluted extraction method provided bythe volatile organic components is that significant concentration of thesample target analyte may be accomplished rapidly and easily in acommercially available sample concentrator within a period of less than3 hours and preferably in less than about one and one-half hours. Thisrapid concentration provided by the volatile organic components rendersthe preferred extraction method of the invention readily adaptable forcommercial laboratory applications.

After the sample has been concentrated to about one-half a milliliter involume, a portion of the sample is preferably reconstituted in a ratiofrom about 0.1:1 to about 1:1 with a 50/50 methanol AD_(x) phosphatebuffer solution to provide a stabilized, buffered concentrated extract.The buffered extract concentrate may be directly tested in commerciallyavailable fluorescence polarization immunoassay equipment such as thepreferred ABBOTT AD_(x) ® analyzer instrument available from AbbottLaboratories. In accordance with an especially preferred aspect of thecurrent method, the concentrated buffered extract will be furthertreated to remove lipids and neutral fats contained therein prior toFPIA screening. The lipids were needed to transfer all target analytefrom the meconium into the extract but after target analytes areseparated therefrom, the residual lipids if left in the buffered extracttend to interfere with subsequent immunoassay screening procedures. Inaccordance with this aspect of the method the neutral fats, fatty acidsand triglycerides may be removed from the buffered concentrated extractby transferring the buffered extract to a 1.5 ml. micro-centrifuge tubeand thereafter centrifuging at high speed for the time sufficient toseparate an organic fatty layer from a buffered target analyteconcentrate. Thereafter, the fatty lipid layer may be removed by anysuitable method including careful aspiration being careful not to removebuffered extract. The remaining de-lipidized or clarified bufferedmeconium extract concentrate, prepared in accordance with the new andimproved method herein, is thereafter analyzed for cocaine metabolite(benzoylecgonine), phencyclidines (PCP), opiates, amphetamines orcannabinoid metabolites using fluorescence polarization immunoassaymethods on Abbott's AD_(x) analyzer, in a manner more particularly to beset forth in the Examples.

In accordance with an important feature of the preliminary immunoassayscreening, it has been found in practice that the internal controls andcalibrators used to determine heroin, morphine or codeine use duringpregnancy should employ free-morphine and codeine forms of the drug asthe internal controls, rather than any metabolite form such as aglucurinide form. This finding is based on the discovery that whenmorphine screening procedures were run, assaying for glucurinide formsand free drug forms in fetal meconium, at least about 90% of the opiatedrugs were present in the free drug form rather than in the glucurinideor metabolized form. Prior art methods which assayed for opiates such asmorphine and codeine assumed that the drug was present in theglucurinide form. The prior methods included an enzyme hydrolysis stepor a high-temperature acid hydrolysis step to convert target analyteprior to assay, and glucurinide forms of the drugs were used as thespiked controls. Applicant has discovered that the prior art methodswere using the wrong comparative standards as internal controls. Inaccordance with this finding, a major time-consuming and expensivehydrolysis step may now be avoided. In accordance with this preferredembodiment of the method, an assay for opiates is performed employing asthe internal control standards, free morphine and free codeine drugs toprovide an improved immunoassay.

The new and improved preliminary screening extraction and immunoassaymethods in accordance with the preferred embodiment of the presentinvention provides to improved sensitivity and detection of targetanalytes in meconium samples compared with prior art methods includingan aqueous acid extraction steps. The volatile organic extractionmethods of this invention when compared to the prior art aqueous acid ormethanol extraction procedure, such as that taught by Ostrea et al.,showed more than ten-fold improvement in detection sensitivity for thenew and improved extraction method of this invention. In an assay screenfor opiates employing free drug as the internal controls and using anFPIA immunoassay procedure in accordance with the method of thisinvention, a screening cut-off concentration or lower limit of opiatedetection in the meconium sample was 50 nanograms of opiate per gram ofmeconium tested. The corresponding lower detection limit for the OstreaJr. method for detecting opiates by screening immunoassay was 600nanograms per gram. Moreover, the preliminary screening cut-off valuesfor detecting phencyclidine (PCP) target analytes was 50 nanograms pergram in accordance with the present method, as compared to a 250nanograms per gram cutoff employing the Ostrea prior art method. Forcannabinoid metabolites, the lower detection limit of the currentinvention pre-screening assay was 50 nanograms per gram, whereas theOstrea Jr. method for cannabinoid metabolites had a lower detectionsensitivity of 500 nanograms per gram. For cocaine metabolite(benzoylecgonine), the lower limit of detection for the preliminaryextraction and screening method in accordance with this invention was100 nanograms per gram of meconium tested, as compared with 500nanograms per gram in the Ostrea Jr. prior art. Similarly, a preliminaryscreening procedure in accordance with the preferred methods of thisinvention was able to detect amphetamines in the patient samples atconcentrations as low as 250 nanograms per gram, whereas the Ostrea Jr.closest prior art methods had a lower detection limit of 1,000 nanogramsper gram.

In accordance with this invention preliminary screening extraction andimmunoassay methods are described for quickly and rapidly identifyingthose neonatal patients whose meconium samples indicate have beengestationally exposed to addictive drugs in utero.

In accordance with the method of this invention, once the preliminaryscreening immunoassay has determined that a sample qualitativelycontains a target analyte being assayed for, whether the target analytebe a drug of abuse or its metabolite, or whether it be some otherchemical agent of interest, another aliquot portion of that sample issubjected to a more precise GC/MS confirmatory procedure tounequivocally identify and quantitate the amount of that specific targetanalyte present in that meconium sample in forensically acceptableterms.

In general terms, the GC/MS confirmatory procedures instead of employinga group extraction approach provide a specific extraction known to bethe best extraction procedure for that particular target analyte. Thesecond aliquot portion of pre-processed meconium is selected as astarting material for the GC/MS confirmatory for a particular analyte.The test sample is spiked with known quantities of labelled analyteanalog. The labelled analyte analogs are preferably deuterated forms ofthe same drug which by virtue of their isotopic deuterated form have aslightly different and identifiable ionic weight in the GC/MS procedure.The spiked sample containing added internal controls in known quantitiestogether with the unknown quantity of target analyte are thereafterextracted by a specific procedure known to be the most effective forthat particular analyte to provide an extract fluid. That extract fluidis separated from meconium solids and purified in a specific procedureto provide a new test fluid containing the spiked internal controlsample. This purified fluid is then derivatized with a trimethylsilylreagent or a heptafluorobutyration reagent to derivatize the spikedinternal controls and sample analytes to machine identifiable derivativeform. Analysis of the derivatized fluid is performed in GC/MS equipmentusing electron impact and selected ion monitoring mode.

As has been mentioned above, the particular GC/MS confirmatory assay isspecific to a given target analyte. More particularly, the confirmatoryGC/MS for cocaine metabolite, benzoylecgonine, improves upon theRosenzweig method mentioned above by starting with a pre-processedmeconium starting material to provide an improved time-averaged aliquotsample. The cocaine confirmatory procedure employs the volatile organicacid and volatile organic solvent co-extraction procedure outlined forthe "cocktail" extract to maximize recovery of benzoylecgonine targetanalyte and the spiked deuterated internal working standards togetherwith some neutral lipid components from the meconium aliquot sample.After the acetic acid/acetone extraction reagents are added, a two-phasemixture is formed and vortexed. The contacted two-phase solution iscentrifuged at high speed to separate the organic layer from the secondlayer. The resulting concentrated non-aqueous meconium extract preparedis reduced in volume in a sample evaporator to 0.5 ml. Thereafter, theconcentrated analyte analogs and target analytes for cocaine arereconstituted in a half a milliliter of ethanol and 6 ml. of a phosphatebuffer at a pH of 6. This mixture is vortexed thoroughly and centrifugedat high speed until a top lipid layer is formed. The top lipid layer isseparated from the lower buffered extract layer by careful aspiration.To this point in the extraction procedure, an improved recovery over theRosenzweig method is accomplished by using the more efficient aceticacid/acetone extraction to recover target analyte still dissolved in thefatty components of the meconium. After separating the target analytesand the unwanted fats together as a unit, the volatiles are driven offin a concentration step. Reconstituting with methanol provides aselective solvent for the cocaine target analytes and analyte analogs inwhich the fats are not particularly soluble. The addition of thephosphate buffer followed by centrifuging now separates the enhancedrecovery of the desired target analytes and analogs, and from theundesired lipid components. This effectively increases the total amountof target analyte recovered from the meconium sample than would beobtained from following the Rosenzweig method of extracting withmethanol alone, as the first step.

The clarified lipid-free buffered extract is subjected to solid phaseextraction using a BOND ELUTE CERTIFY brand (VARIAN) solid phaseextraction column by sequentially passing through each column, methanol,phosphate buffer at pH 6 and sample. After rinsing, selectivelyextracted target analytes and analyte analogs are eluted with a polarorganic solvent containing ammonium hydroxide. The resulting eluate istaken to dryness in a sample concentrator and reconstituted in alcohols.Thereafter the trimethylsilyl derivative is made by incubating theeluted target analytes with the trimethylsilyl derivatizing agent withheating for a time sufficient until derivatization is complete.Thereafter, the derivatized sample is injected into the gaschromatograph/mass spectrometer.

The amount of cocaine metabolite target analyte present in the meconiumsample is calculated based on a comparison of the native cocaine andbenzoylecgonine sample response to the known response of thequantitatively known deuterated cocaine and benzoylecgonine analogs. Theratio of the target analyte area versus the labelled analog area isdetermined and this ratio is multiplied by the amount of labelled analogadded to the extract divided by the mass of the meconium sample tested.The resulting calculation expresses the quantity of cocaine and/orcocaine metabolite present in the original sample in terms of nanogramsof cocaine or metabolite per gram of meconium tested.

The new and improved GC/MS confirmatory assay for cocaine and itsmetabolite, benzoylecgonine, in accordance with this invention is ableto detect analyte concentrations greater than or equal to 25 nanogramsper gram. The closest prior art confirmation has a corresponding lowerdetection sensitivity or limit of 250 nanograms per gram. The new andimproved cocaine confirmatory procedure of this invention exhibited aten-fold improvement in detection sensitivity over the prior art.

In accordance with the present invention, heretofore unavailable GC/MSconfirmatory methods for quantifying the amount of opiate analytes,amphetamine analytes, tetrahydrocannabinoid metabolite analytes andphencyclidine analytes in neonatal meconium samples are now provided. Inaccordance with these new confirmatory procedures for opiates and foramphetamines, each employ a specialized liquid/liquid extractiontechnique is employed for each target analyte. In the opiateconfirmation assay, a sample of pre-processed meconium is spiked withknown quantities of deuterated codeine and morphine analogs as internalstandard controls. Thereafter, the spiked meconium sample is extractedby homogenizing in concentrated hydrochloric acid to release targetanalytes from the meconium matrix. After being homogenized andcentrifuged, the top aqueous acid layer is transferred to a silanized,capped glass tube to provide a first aqueous acid extract. In the opiateconfirmation assay, the first acid extract is extracted withmethyl-t-butyl ether to remove lipids and the top organic layer iscarefully aspirated and discarded. The clarified acid layer isneutralized with base and then back extracted into an organic ethersolvent and buffered. The buffered extract is centrifuged and the toporganic phase is removed to provide a final opiate extract forconcentration in a sample concentrator. The samples are taken to drynessin the sample concentrator, reconstituted in ethanol, transferred toauto-sampler vials and taken to dryness at elevated temperature andreduced pressure. The target analytes and opiate analogs present in thedried auto-sampler vial are derivatized by vapor phase derivatizationwith a N-methyl-N-trimethylsilyltrifluoroacetamide derivatizing agent.Thereafter the GC/MS spectra is taken in selective ion mode. Thequantity of opiates in the form of morphine or codeine present in theoriginal meconium sample is determined by comparing the native opiatedrug response against the response of the quantitatively knowndeuterated analogs. A ratio of the native drug area versus the labelledanalog area in the extract multiplied by the concentration of thelabelled analyte in the extracted mass yields a final quantitativemeasurement of opiate concentration in terms of nanograms of opiate pergram of meconium tested.

In the amphetamine confirmation assay a sample of pre-processed meconiumis spiked with working internal standards of d-amphetamine-d5 andmethamphetamine-d8 to provide a spiked meconium sample. The spikedsample is extracted with concentrated hydrochloric acid withhomogenization followed by centrifugation to define a top aqueous acidlayer. The aqueous acid layer extract is also transferred to a silanizedglass tube for further processing. In the amphetamine confirmation, thefirst acid extract is neutralized with base, buffered and extracted in amixed organic solvent extraction into a top organic solvent phase. Thetop organic phase is transferred to a clean tube, re-acidified withhydrochloric acid and centrifuged to define a top organic phase and alower aqueous acid phase containing target analyte and spikedamphetamine analogs. Once again, the acid extract solution isneutralized, buffered and back extracted with an organic solvent tocollect the amphetamine target analyte and analog values into theorganic phase. After centrifugation and separation, the top organicphase is transferred to an aluminum concentrator vial, treated with asulfating agent and taken to dryness with heat and forced air. Thesample analytes are reconstituted in ethyl alcohol, transferred to anauto-sampler vial and again taken to dryness at elevated temperature andreduced pressure. The target analytes are reconstituted in an organichydrocarbon solvent and derivatized with anN-methyl-bis(heptafluorobutyramide) derivatizing agent. The derivatizedsolution is run through the GC/MS in selective ion monitoring mode. Theamount of amphetamines contained in the original meconium product isdetermined by comparing the native amphetamine response against theresponse of the spiked amphetamine analogs. The ratio of native drugarea versus the labelled analog area times the analog concentration inthe extracted mass yields a final quantitative determination ofamphetamine present in terms of nanograms of amphetamine per gram ofmeconium sample tested.

The new and improved confirmatory procedure for quantitativelydetermining the amount of marijuana metabolite found in a meconiumsample comprises a selected assay for11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (thedelta-9-carboxy-THC or glucurinide metabolite). In accordance with thisprocedure, the deuterated glucurinide metabolite is spiked into a sampleof pre-processed meconium. In practice, it has been shown that aceticacid/acetone extraction for the glucurinide metabolite of marijuana isnot as efficient an extraction agent as absolute methanol. Accordingly,for this marijuana metabolite confirmatory assay, methanol is used asthe extraction solvent. After spiking and adding methanol, the sample ishomogenized for a time period and allowed to rest for five minutes. Aconcentrated base in the form of sodium or potassium hydroxide is addedand the mixture is vortexed. The vortexed mixture is thereaftercentrifuged and the top phase is transferred to a clean, silanized glasstube. The top phase is diluted with de-ionized water and an extractionsolvent including ethyl acetate and N-hexane. The second mixture isvortexed and permitted to incubate for 15 minutes. The top organic phaseis aspirated and discarded. Concentrated HCL is added to each tube andvortexed to convert the target analytes and analyte analogs to theirorganic soluble form. A fresh organic extraction solvent is added to thetube to form a two-phase extraction mixture. After thorough mixing andseparation by centrifuge, the top organic phase is transferred toconcentration cups. The purified organic extracts are taken to drynessin a sample concentrator at elevated temperature and reduced pressure.After reconstituting in alcohol and being transferred to auto-samplervials, the samples are again taken to dryness at elevated temperature ina vacuum. Thereafter, the target analytes and analogs are derivatizedwith a N-methyl-N-trimethylsilyltrifluoroacetamide derivatizing agent.Thereafter, the derivatized sample is injected into the GC/MS andselected ions are monitored. The results are determined by comparing thenative drug response to the quantitatively known deuterated analogspiked into the sample. The quantitative calculation is made bymultiplying the ratio of the native marijuana metabolite area foundversus the area of the deuterated marijuana metabolite analogs. Thatratio is then multiplied by the amount of labelled analog in the extractand divided by the extract mass to provide a final determinationexpressed in terms of nanograms of marijuana metabolite present per gramof meconium tested.

In accordance with still another alternate aspect, the present inventionprovides a new and improved forensically-acceptable GC/MS confirmatoryprocedure for unequivocally identifying and quantifying the presence ofphencyclidine (PCP) analytes in neonatal meconium samples to provematernal use of PCP during pregnancy. More particularly, the PCPconfirmation method of this invention first includes the step of spikinga known amount of deuterated PCP analog into a known quantity ofpre-processed meconium to form a spiked sample. The spiked sample ishomogenized with glacial acetic acid until a substantially uniformhomogenate is obtained. Acetone and diphenylamine are added to form atwo-phase mixture which is vortexed then centrifuged to define a toporganic phase and a second phase. The top organic is transferred to asilanized concentration cup and a minor amount of alcoholic sulfuricacid is added to prevent target analyte from entraining with evaporatingvolatile organic solvents. The transferred organic phase containingtarget analytes and analyte analogs is concentrated at elevatedtemperature and with reduced pressure until the volume approaches about0.5 ml. The concentrated analyte phase is reconstituted in methanol andtransferred to a polypropylene tube. The reconstituted concentrate isbuffered, vortexed and centrifuged. Any top lipid layer is carefullyremoved by aspiration and discarded. The remaining clarified andbuffered extract is passed through a BOND ELUTE CERTIFY brand solidphase extraction column. After column rinsing, the phencyclidine targetanalytes and deuterated analyte analogs are eluted with ammoniumhydroxide and ethyl acetate. The eluate is placed in an aluminumconcentration vial and evaporated to dryness.

The residue is thereafter reconsitituted reconstituted with a mixedIsooctane/isopropanol (80/20) solvent and carefully transferred toauto-sampler vials containing 250 microliter volume inserts. The sampleis injected into the GC/MS instrument and read. The amount ofphencyclidine present is determined by comparing the ratio of theresponse area for sample analyte to the area of the response for thespiked analyte analog. Thereafter, the response ratio is multiplied bythe amount of known spiked internal control over the mass of the sampleto provide the quantity of phencyclidine present in the meconium samplein terms of nanograms of phencyclidine per gram of meconium tested.

Further details and advantages provided by the present invention, in allits aspects, will become apparent from the following examples.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Preparation A--Silanization ofGlassware

Glass, by its chemical nature, has a very active surface that containsmany free hydroxyl groups. One of the disadvantageous characteristics oflaboratory glassware is the ability of the glass surfaces to bindorganic compounds such as drug analytes and their metabolites. Toovercome this error-introducing problem, the glass surfaces may bedeactivated by the formation of silyl ethers with the free surfacehydroxyl groups. This is accomplished in the vapor phase by reacting theglass surfaces with dimethyldichlorosilane (DMCS). In accordance withthe preferred embodiment of this invention, all glassware which comes incontact with the various samples and sample extracts and solutions willbe silanized in accordance with the following procedure.

In accordance with this preparatory method, glassware to be used andtreated is placed in an appropriate rack. A 2 ml vial containing atleast about 1 ml of dimethyldichlorosilane (DMCS) is placed into avacuum oven set at elevated temperatures of between about 75° to 85° C.The rack and glassware are placed into the vacuum oven and the door istightly closed. The vacuum valve is opened to initiate vacuum in theoven. When the vacuum gauge indicates an internal oven pressure of lessthan or equal to 25 inches of mercury, the vacuum valve is closed. Theglassware is permitted to react with the silanizing agent vapors for atleast about 15 minutes. Thereafter, the vacuum valve is opened and theDMCS vapors are exhausted from the vacuum oven. The glassware is removedfrom the oven and allowed to cool to room temperature. All of thetreated glassware is thereafter rinsed in clean reagent grade acetone.After rinsing, the glassware is permitted to thoroughly dry prior touse.

The silanized treated glassware should be clear of any signs ofcontamination and feels slightly slippery to the touch. Any glasswarethat is discolored or smudged or which lacks a slippery feel to thetouch should not be used.

Preparation B--Pre-Processing of the Pooled Meconium Sample

A meconium sample is prepared for extraction and testing by collectingeach of the meconium bowel movements produced by a neonatal infant inthe immediate postpartum period in a polyethylene-lined diaper. Themeconium retained on the polyethylene liner sheet placed in each diaperis transferred to a polystyrene sample container equipped with ascrew-on lid. Between collections, the pooled sample is kept underrefrigeration until sample collection is substantially complete, e.g.,typically after the first two to three days of the neonate's life. Themeconium excreta is collected and pooled until the first appearance ofthe transitional stool is observed. Transitional stool is not includedin the sample specimen.

When sample collection is substantially complete, the pooled meconiumexcreta is pre-processed by introducing a flat bladed instrument, suchas a wooden spatula, into the sample vial and vigorously mashing andmixing the meconium matrix in an effort to remove obvious striationspresent in the meconium tissue samples and to achieve a redistributionof the target analytes within all of the collected meconium. The effortis to try and achieve a smoothed, substantially uniform, pre-processedproduct so that the aliquot portions taken from the pre-processedproduct will exhibit low sample to sample variability- The meconiumtissue is extremely rubbery and resists being smoothed. However,improved reproducibility and accuracy for the testing requires that thepre-processing be as thorough, complete and effective as possible. Inany event, some pre-stirring pre-processing is certainly better thannone and vigorous stirring, mashing and mixing for a period of from 1 to5 minutes with the bladed spatula is generally effective to create thesmooth, evenly-distributed product.

EXAMPLE 1 Method for Making Concentrated Cocktail Meconium Extract forPreliminary Screening by Fluorescence Polarization Immunoassay Methods

A non-aqueous, substantially non-diluted, concentrated volatile organiccocktail extract was prepared as follows:

A number of reagent solutions were prepared prior to performing theextraction. A volatile organic solvent including a minor effectiveamount of a glass anti-binding agent was prepared by weighing 6.68milligrams of diphenylamine and adding it to a fresh 4 liter bottle ofreagent grade acetone. After thorough mixing, the 1.67 mg/ldiphenylamine in acetone solution was labelled.

A stable buffer comprising a 50/50 volume/volume mixture of ABBOTTAD_(x) ® buffer in absolute methanol was prepared by mixing equalportions of the AD_(x) buffer and methanol together in a suitably sizedcontainer. The AD_(x) buffer/MeOH reagent solution was labelledappropriately.

A 0.1% H₂ SO₄ in MeOH solution was prepared by pipetting 100 microlitersof concentrated sulfuric acid into 100 milliliters of absolute methanol.The alcoholic sulfuric acid solution was labelled appropriately andstored for a period of up to one month.

After preparation of the reagents, the new and improved cocktail extractmethod was performed in accordance with this invention as follows:

A 1.0 gram sample of the pre-processed neonatal meconium obtained inPreparation B set forth above was placed in a 16 by 125 polypropylenetube. 3.0 mls. of reagent grade glacial acetic acid was added to thepolypropylene tube and sample. The volatile organic acid andpre-processed meconium were homogenized using an OMNI 5000 tissuehomogenizer set at the highest speed for a period of at least about 15seconds until a smooth, substantially uniform finely-divided homogenatewas formed. The homogenate after the tissue homogenizing treatment hadan average particle size of between about 2 to about 5 microns. Afterhomogenization, 6.0 mls. of the acetone/diphenylamine reagent solutionwas added to the sample tube. The resulting mixture was vortexed andallowed to mix in a sample mixer for more than 5 minutes. The resultingtwo-phase mixture including an organic phase and a second phaseincluding the meconium solids and other materials is separated bycentrifuging at high speed for a period of about 10 minutes. The toporganic acetone phase is decanted through a 30 micron polypropylene fritand added to a silanized concentration cup. A drop of the 0.1% H₂ SO₄ inmethanol solution was added to each cup, to prevent loss of amphetamineanalyte due to co-evaporation with entrained solvents. The resultingorganic extract was concentrated to a volume of less than 0.5 ml. in anAlltech sample concentrator, Model 190-A set at 75° C. over air andvacuum. The resulting concentrated sample appeared to have a top lipidlayer with little or no sample volume thereunder. After concentration,the target analytes are reconstituted with 0.7 ml. of the AD_(x)buffer/MeOH reagent. In accordance with a preferred feature of thisinvention, the interfering fats and lipid phases are removed from thereconstituted buffered extract by transferring the reconstituted extractto a 1.5 ml. microcentrifuge tube and centrifuged at high speed for aperiod of at least about 5 minutes. The top organic lipid layer wascarefully aspirated and removed to provide a clarified, concentrated andbuffered meconium extract for further testing.

EXAMPLE 2 Qualitative Preliminary Screening Procedure for CocaineMetabolite

An aliquot portion of the clarified, buffered meconium extractconcentrate prepared in accordance with the method of Example 1 wasevaluated for the presence of cocaine metabolite (benzoylecgonine) by acommercially available fluorescence polarization immunoassay (FPIA)method on commercially available ABBOTT AD_(x) ® analyzer equipmentemploying the AD_(x) instrument its protocols and ABBOTT AD_(x) ®reagents.

The FPIA cocaine metabolite assay is a homogeneous fluorescencepolarization immunoassay designed to detect the cocaine metabolitebenzoylecgonine in biological specimens. The assay is based oncompetition between any cocaine metabolite present in the sample andbenzoylecgonine labelled with fluorescein for a limited number ofantibody binding sites. Fluorescence polarization decreases upon bindingof the fluorescein labelled benzoylecgonine to the antibody, and theamount that is bound is inversely related to the benzoylecgonineconcentration in the sample. Thus, in accordance with the instrumentalmethod, fluorescence polarization decreases directly withbenzoylecgonine concentration present in the sample. The degree offluorescence polarization is measured by a liquid crystal in theinstrument and the concentration of immuno-reactive benzoylecgonineequivalence is determined on the ABBOTT AD_(x) ® analyzer comparing theinstrument value of the unknown with a standard curve derived from theinstrument values of known calibrators and internal controls.

An ABBOTT AD_(x) ® cocaine metabolite assay kit (ABBOTT Catalog Number9670-77) is equipped with a number of assay reagents and a number ofcalibrators comprising human urine spiked with benzoylecgonine atcertain known concentrations. For purposes of this new and improvedmeconium assay procedure certain negative control solutions are preparedin preparation for performing the FPIA immunoassay. A Lyphocheck screencontrol containing 375 nanograms per milliliter of benzoylecgonine wasobtained from BIO-RAD, INC. under their Catalog Number 476 in 10, 10 ml.samples. The unopened bottles were stored at 2° to 8° C. prior to use.Before performing the assay, the screen controls from BIO-RAD werepermitted to equilibrate to room temperature. The control solution wasswirled to insure homogeneity being careful to avoid foaming.

A negative control sample was obtained from UNITED STATES DRUG TESTINGLABORATORIES. The negative control samples were prepared in 500 ml.bottles and are certified to be drug-free. These negative controlsamples are stored under refrigeration at 2° to 8° C. when not in use.The negative controls obtained from USDTL were not used and discarded ifbacterial contamination or turbidity was evidence upon visualinspection.

Negative meconium controls were prepared by weighing 1 gram samples ofdrug-free meconium and performing the extraction procedure set forth inExample 1 to provide the negative control fluid for use in the FPIAassay.

Positive meconium controls were prepared by taking 1 gram samples ofdrug-free meconium and pipetting 100 microliters each of a spikingsolution having a benzoylecgonine concentration of 4,000 nanograms perml. and a cocaine control spiking solution containing 2,000 nanogramsper ml. of cocaine, respectively.

After preparation of all the calibrators and controls and equilibrationto room temperature was substantially completed, a 100 microliter sampleof the cocktail extract solution obtained in accordance with the methodof Example 1 is employed as the sample unknown for FPIA analysis for thepresence of cocaine metabolites in the original meconium sample. TheFPIA immunoassay sample was run in accordance with standard ABBOTTAD_(x) ® procedures. The instrument values were recorded. A meconiumextract sample unknown that gives an instrument derived value greaterthan the stored threshold value (in part determined by the negativecontrols) was recorded as a positive finding for cocaine metabolite inthat sample.

Repeated experiments against known positive control specimens employingthe methods of Examples 1 and 2 has revealed that the qualitative FPIApreliminary screening assay in accordance with this invention forcocaine metabolite is capable of providing reproducibly accuratepositive determinations of the presence of cocaine metabolite atconcentrations of benzoylecgonine as low as 15 nanograms per gram ofmeconium sample tested.

EXAMPLE 3 Qualitative Preliminary Screening Procedure for AmphetamineClass Analytes

An aliquot portion of the clarified buffered meconium extractconcentrate prepared in accordance with the method of Example 1 wasevaluated for the presence of d-amphetamine or methamphetamine by anABBOTT AD_(x) ® FPIA immunoassay in a manner similar to the method ofExample 2. The FPIA amphetamine class analytes assay is a homogeneousfluorescence polarization immunoassay designed to detectd,l-amphetamine, d-amphetamine and methamphetamine in biologicalspecimens. Other structurally similar phenethylamines can also producepositive results. The assay is based on competition between anyamphetamines present in the sample and amphetamine labelled withfluorescein for a limited number of antibody binding sites. Fluorescencepolarization decreases upon binding of the fluorescein labelledamphetamine to the antibody, and the amount that is bound is inverselyrelated to the amphetamine concentration in the sample. Accordingly,fluorescence polarization decreases directly with amphetamineconcentration. The degree of fluorescence polarization is measured by aliquid crystal in the instrument and the concentration of theimmunoreactive amphetamine equivalents is determined on the ABBOTTAD_(x) ® analyzer by comparing the instrument value of the amphetaminetarget unknowns with a standard curve derived from the instrument valuesof known calibrators and internal controls.

An ABBOTT AD_(x) ® amphetamines class assay kit (ABBOTT Catalog Number1A99-77) is equipped with a number of assay reagents and a number ofcalibrators comprising human urine spiked with d-amphetamine at certainknown concentration. For purposes of this new and improved meconiumassay procedure, certain negative control solutions are prepared inpreparation for performing the FPIA assay. A Lyphocheck screen controlcontaining 1,250 n/ml. amphetamine was obtained from BIO-RAD, INC.,under their Catalog Number 476 in 10, 10 ml. vials. The unopened bottlesare stored at 2° to 8° C. prior to use. Before performing the assay, thescreen controls from BIO-RAD are permitted to equilibrate to roomtemperature. The BIO-RAD control solutions are swirled to insurehomogeneity with care being taken to avoid foaming.

A negative control sample was obtained from UNITED STATES DRUG TESTINGLABORATORIES. The negative control samples were prepared in 500 ml.bottles and were certified to comprise drug-free human urine. Thesenegative control samples were stored under refrigeration at 2° to 8° C.when not in use. The negative controls obtained from USDTL were notused, but were discarded if bacterial contamination or turbidity wasevident upon visual inspection.

A negative meconium control sample was prepared by weighing a 1.0 gramsample of drug-free meconium and performing the extraction procedure asset forth in Example 1 to provide the negative control fluid extract foruse in the FPIA assay.

Positive meconium controls were prepared by spiking 1.0 gram samples ofdrug-free meconium with 100 microliter aliquots of spiking solutionscontaining 1,000 n/ml amphetamine and 1,000 n/ml. methamphetamine,respectively.

After preparation of all the calibrators and controls and equilibrationto room temperature, a 100 microliter sample of the cocktail extractsolution obtained in accordance with the method of Example 1, isemployed as the sample unknown for FPIA analysis for the presence ofamphetamine in the original meconium sample. The FPIA immunoassay samplewas run in accordance with standard ABBOTT AD_(x) ® procedures. Theinstrument values were recorded. A meconium extract sample unknowngiving an instrument derived value greater than the stored thresholdvalues (in part determined by the negative controls) was recorded as apositive finding for amphetamine in that sample.

Repeated experiments against known positive control specimens employingthe methods of Examples 1 and 3 herein has revealed that the qualitativeFPIA preliminary screening assay in accordance with this invention foramphetamine is capable of providing reproducibly accurate positivedeterminations of the presence of amphetamine at concentrations ofamphetamine or methamphetamine as low as 250 ng/g. of sample meconiumtested.

EXAMPLE 4 Qualitative Preliminary Screening Procedure for OpiateAnalytes

An aliquot portion of the clarified, buffered meconium extractconcentrate prepared in accordance with the method of Example 1 wasevaluated for the presence of morphine and codeine by an ABBOTT AD_(x) ®FPIA immunoassay in a manner similar to the method described in Examples2 and 3. The FPIA opiate assay is a homogeneous fluorescencepolarization immunoassay designed to detect morphine and codeine inbiological specimens. Other structurally similar opiates can alsoproduce positive results. The assay is based on competition between anyopiates present in the sample and morphine labelled with fluorescein fora limited number of antibody binding sites. Fluorescence polarizationdecreases upon binding of the fluorescein labelled morphine to theantibody, and the amount that is bound is inversely related to themorphine concentration in the sample. Thus, fluorescence polarizationdecreases directly with morphine concentration. The degree offluorescence polarization is measured by a liquid crystal and theconcentration of immunoreactive morphine equivalents is determined onthe ABBOTT AD_(x) ® analyzer comparing the instrument value of theunknown against a standard curve derived from the instrument values ofknown calibrators and internal controls.

An ABBOTT AD_(x) ® opiate assay kit (ABBOTT Catalog Number 9673-77) isequipped with a number of assay reagents and spiked human urinecalibrators including known quantities of morphine. For purposes of thisnew and improved meconium assay procedure, certain negative controlsolutions were prepared in preparation before performing the FPIAimmunoassay. A Lyphocheck screen control solution containing 375 n/ml.of morphine obtained from BIO-RAD, INC. under their Catalog Number 476in 10 ml. vials were kept in stored unopened bottles under refrigerationuntil ready for use. Immediately before use the known control sampleswere permitted to equilibrate to room temperature and gently swirled toinsure homogeneity.

A negative control sample comprising drug-free urine was obtained fromthe UNITED STATES DRUG TESTING LABORATORIES. A negative meconium controlwas prepared by weighing a 1.0 gram sample of certified drug-freemeconium and subjecting that sample to the extraction procedure setforth in Example 1 to provide a negative control fluid for use in theFPIA assay. Positive meconium controls were prepared by taking 1.0 gramsamples of drug-free meconium and spiking them with 100 microliters of aspiking solution containing 1,000 n/ml. of morphine and codeine,respectively. After preparation of all calibrators and controls, alltest solutions were permitted to come to room temperature and a 100microliter sample of the cocktail extract solution obtained inaccordance with the method of Example 1 was employed as the sampleunknown for FPIA 25 analysis for the presence of opiates in the originalmeconium sample. The FPIA immunoassay sample was run in accordance withstandard ABBOTT AD_(x) ® protocols in accordance with the methoddescribed in Examples 2 and 3.

Repeated experiments against known positive control specimens employingthe method of Example 4 has revealed that the qualitative FPIApreliminary screening assay in accordance with this invention foropiates is capable of providing reproducibly accurate positivedeterminations of the presence of opiate analytes at concentrations ofmorphine or codeine as low as 50 ng/g. of meconium sample tested.

EXAMPLE 5 Qualitative Preliminary Screening Procedure for CannabinoidAnalytes

An aliquot portion of the clarified, buffered meconium extractconcentrate prepared in accordance with the method of Example 1, wasevaluated for the presence of marijuana metabolites by an ABBOTT AD_(x)® FPIA immunoassay in accordance with the methods of Examples 2-4.

The cannabinoid delta-9-tetrahydrocannabinol-(delta⁹ -THC) is theprinciple psychoactive ingredient marijuana and hash-hish. The compounddelta⁹ -THC is quickly and effectively absorbed by inhalation or fromthe gastrointestinal tract and is almost completely metabolized.Excretion of urinary metabolites begins within hours after exposure tocannabinoids. The prevalent theory is that delta⁹ -THC is distributed inand absorbed by various fatty tissues and then is very slowly releasedto the plasma. Thereafter, it is readily metabolized in the liver andeventually excreted in the urine and feces. The ABBOTT fluorescencepolarization cannabinoid assay detects the major metabolite of delta⁹-THC, i.e. 11-nor-delta⁹ -THC-9-carboxylic acid. It also detects otherdelta⁹ -THC metabolites.

The FPIA assay is a homogeneous fluorescence polarization immunoassaytechnique used for the analysis and detection of cannabinoid metabolitecompounds in biological fluids. As with the other ABBOTT AD_(x) ®assays, the assay is based on competition between drug metabolitespresent in the sample and known quantities of fluorescein labelled drugmetabolite analogs added to the sample specimen which compete for aknown quantity of antibody binding sites.

For this marijuana metabolite assay the ABBOTT AD_(x) ® cannabinoidsassay kit (ABBOTT Catalog Number 9671-77) was employed. In addition tothe standard cannabinoid assay reagents provided with the ABBOTT kit, anumber of internal open controls were prepared especially in accordancewith the methods of the invention. More particularly, a Lyphocheckscreen control containing 125 n/ml. of cannabinoid was obtained fromBIO-RAD, INC. under their Catalog Number 476 in a quantity of 10, 10 ml.vials. A negative control of drug-free urine was obtained from UNITEDSTATES DRUG TESTING LABORATORIES in 500 ml. bottles.

Negative meconium controls were also prepared by extracting 1.0 gramsamples of known drug-free meconium in accordance with the extractionmethod set forth in Example 1. Positive meconium controls were preparedby spiking 1.0 gram samples of drug-free meconium with 100 microliteraliquot of a solution containing 580 n/ml. of THC-COOH glucurinide.

A 100 microliter sample of the cocktail extract solution obtained inaccordance with the method of Example 1 was employed as the sampleunknown for the FPIA analysis for the presence of marijuana metabolitein the original meconium sample. The FPIA immunoassay was run inaccordance with standard ABBOTT AD_(x) ® protocols. The instrumentvalues were recorded and any instrument values greater than storedthreshold values were recorded as a positive finding for marijuanametabolites in the sample.

Repeated experiments against known positive control specimens employingthe methods of Examples 1 and 5 has revealed that the qualitative FPIApreliminary screening assay in accordance with this invention formarijuana metabolite is capable of providing reproducibly accuratepositive determinations of the presence of marijuana metabolite atconcentrations of the glucurinide metabolite as low as 50 ng/g. ofmeconium sample tested.

EXAMPLE 6 Qualitative Preliminary Screening Procedure for Phencyclidine(PCP) Analytes

An aliquot portion of the clarified, buffered meconium extractconcentrate prepared in accordance with the method of Example 1 wasevaluated for the presence of PCP by an ABBOTT AD_(x) ® FPIA immunoassayin a manner similar to the method of Examples 2-5. The FPIAphencyclidine analyte assay is a homogeneous fluorescence polarizationimmunoassay designed to detect PCP in biological specimens. An ABBOTTAD_(x) ® phencyclidine-2 assay (ABBOTT Catalog Number 9672-76) wasemployed as the reagent and calibration kit for the assay. Additionalcontrols including a Lyphocheck screen control containing 32 n/ml. ofPCP obtained from BIO-RAD, INC. under BIO-RAD Catalog Number 476 in 10ml. sample vials was obtained. Negative control urine samples fromUNITED STATES DRUG TESTING LABORATORIES in 500 ml. bottles was alsoobtained. Negative meconium control samples comprising 1.0 gram samplesof drug-free meconium extracted in accordance with the method of Example1 to provide a negative meconium control test fluid were also prepared.Positive meconium controls were prepared by spiking 1.0 gram samples ofknown drug-free meconium with 100 microliters of a PCP solutioncontaining 500 n/ml. PCP. The FPIA analysis employing a 100 microlitersample of the cocktail extract solution obtained in accordance with themethod of Example 1 was run on the analyzer employing standard ABBOTTAD_(x) ® procedures.

Repeated experiments against known positive control specimens employingthe methods of Examples 1 and 6 has revealed that the qualitative FPIApreliminary screening assay in accordance with this invention for PCP iscapable of providing reproducibly accurate positive determinations ofthe presence of PCP analyte at concentrations of PCP as low as 50 ng/g.of meconium tested.

EXAMPLE 7

Comparative Extraction Efficiencies of Meconium Tissue Samples by aVariety of Volatile Organic Acids

In the following examples, experiments were conducted to determine themost efficient volatile organic reagent system for providing maximumcocktail recovery of the various target analytes from a meconium tissuespecimen in a single extraction procedural step. In accordance with thisstudy, six negative drug-free meconium specimens were spiked with knownamounts of several target analytes to form a spiked positive controlspecimens identified as Positive Controls A through F. Each of thespiked positive controls samples A through F were spiked with thefollowing added target analytes:

    ______________________________________                                        TARGET ANALYTE      NG/GRAM                                                   ______________________________________                                        THC-COOH GLUCURINIDE                                                                               58 NG/GRAM                                               BENZOYLECGONINE     400 NG/GRAM                                               COCAINE             200 NG/GRAM                                               CODEINE             100 NG/GRAM                                               MORPHINE GLUCURINIDE                                                                              100 NG/GRAM                                               PCP                  50 NG/GRAM                                               AMPHETAMINE         100 NG/GRAM                                               METHAMPHETAMINE     100 NG/GRAM                                               ______________________________________                                    

Each of these spiked positive control specimen samples were extracted inaccordance with the general method outlined in Example 1 except that forsome aliquot portions of the positive control samples a volatile organicacid selected from acetic acid, propionic acid, trifluoroacetic acid andformic acid were used as the analyte liberating reagents. Thereafter,the volatile organic acetone solvent was added in the extractionprotocol of Example 1 and was followed until a clarified, bufferedmeconium extract concentrate was obtained for each organic acidextracting agent and for each positive control sample. Thereafter,aliquot portions of the clarified, buffered extract concentrates foreach acid and each positive control sample were screened in accordancewith the screening procedures outlines in Examples 2-6 to make aqualitative determination of the amount of spiked analyte present in thespiked positive control samples A through F. The results obtained forthe various volatile organic acid extracting agents for each of thesesix positive control specimens as determined by the ABBOTT AD_(x) ® FPIAprotocols is set forth in Table 1 as follows:

                  TABLE 1                                                         ______________________________________                                        COMPARATIVE EXTRACTION EFFICIENCIES OF                                        VARIOUS ORGANIC ACIDS FOR MECONIUM                                            BOUND TARGET ANALYTES                                                                    THC  COCN    OPIATES   PCP  AMPS                                   ______________________________________                                        ACETIC ACID                                                                   POS CONTROL 1                                                                              67.7   549.0   136.0   19.3 1418.0                               POS CONTROL 2                                                                              66.5   584.0   147.0   29.4 1668.0                               POS CONTROL 3                                                                              68.5   520.0   131.0   17.0 1884.0                               POS CONTROL 4                                                                              61.7   403.0   128.0   17.9 1752.0                               POS CONTROL 5                                                                              59.4   970.0   119.0   22.7 1232.0                               POS CONTROL 6                                                                              55.6   363.0   121.0   18.1 2010.0                               MEAN VALUE   63.2   564.8   130.3   20.7 1660.7                               STD DEV       4.7   197.4    9.4     4.3  265.5                               PROPIONIC ACID                                                                POS CONTROL 1                                                                              64.9   307.0   103.0   12.8 1661.0                               POS CONTROL 2                                                                              74.3   488.0   101.0   19.0 1577.0                               POS CONTROL 3                                                                              59.4   564.0   111.0   42.3 1771.0                               POS CONTROL 4                                                                              62.9   588.0   126.0   53.5 1514.0                               POS CONTROL 5                                                                              69.2   303.0    95.0   13.8 1888.0                               POS CONTROL 6                                                                              77.9   369.0    98.0   31.9 1604.0                               MEAN VALUE   68.1   436.5   105.7   28.9 1669.2                               STD DEV       6.4   116.2    10.4   15.2  125.9                               TRIFLUORACETIC ACID                                                           POS CONTROL 1                                                                              0.0     0.0    70.0    0.0  1170.0                               POS CONTROL 2                                                                              3.4    292.0   86.0    5.0   886.0                               POS CONTROL 3                                                                              0.0     72.0   95.0    0.0  1671.0                               POS CONTROL 4                                                                              2.0    533.0   88.0    1.7   687.0                               POS CONTROL 5                                                                              10.0   351.0   83.0    10.4  951.0                               POS CONTROL 6                                                                              0.0    209.0   96.0    0.0  1578.0                               MEAN VALUE   2.6    242.8   86.3    2.9  1157.2                               STD DEV      3.6    176.8    8.7    3.8   360.2                               FORMIC ACID                                                                   POS CONTROL 1                                                                              18.3   453.0   133.0   25.3 1926.0                               POS CONTROL 2                                                                              21.4   543.0   152.0   32.7 1345.0                               POS CONTROL 3                                                                              20.3   453.0   118.0   34.4 1958.0                               POS CONTROL 4                                                                              23.2   571.0   147.0   30.5 1895.0                               POS CONTROL 5                                                                              25.3   450.0   126.0   25.2 1948.0                               POS CONTROL 6                                                                              22.1   403.0   149.0   26.2 1693.0                               MEAN VALUE   21.8   478.8   137.5   29.1 1794.2                               STD DEV       2.2    58.5    12.7    3.7  219.2                               ______________________________________                                    

From the data of Table 1 the increasing order of extraction efficiencyfor the volatile organic acids in combination with the volatile acetonesolvent extraction system was trifluoroacetic acid<formic acid<propionicacid<acetic acid.

EXAMPLE 8 GC/MS Confirmation Procedure For Quantitative MeconiumAnalysis For Cocaine

In accordance with this invention, neonatal meconium samples testingpositive for cocaine metabolite, benzoylecgonine, in the preliminaryFPIA immunoassay screening procedures described in Examples 1 and 2,were subjected to a confirmatory quantitative GC/MS analysis to providea forensically acceptable determination of the amount of cocaine andbenzoylecgonine present in the meconium sample. Generally, secondaliquot portions of the pre-processed pooled meconium samples for thesesubject infants yielding a presumed positive by FPIA screen were spikedwith trideuterated cocaine and benzoylecgonine as internal standards.The extraction procedure on the spiked pre-processed specimens includedan initial extraction with acetic acid/acetone to provide a cocaineextract concentrate. The cocaine extract concentrate was buffered andcentrifuged to separate neutral fats and lipids prior to furtherprocessing. The clarified extracts were buffered to a pH of 6.0 andsubjected to a solid phase extraction employing a cationic solid phaseextraction column to selectively isolate and separate cocaine andbenzoylecgonine target analytes in a concentrated eluate. The eluate wastreated to form trimethylsilyl derivatives of the spiked analyte analogsand sample analytes and the resulting derivatized solutions wereanalyzed by gas chromatography/mass spectrometry (GC/MS) on aHewlett-Packard GC-MSD instrument.

Before performing the specialized extraction for cocaine and itsmetabolites, the following calibration standards and internal workingcontrols and reagents were prepared:

Calibration Standards: 100 ng each of cocaine and benzoylecgonine spikedwith 500 ng each of deuterated analogs were prepared. Cocaine-d3 andbenzoylecgonine-d3 were used as the internal standards (0.1 ng/ml inmethanol). Working internal standards included codeine, cocaine-d3 andbenzoylecgonine-d3 at 20 micrograms per ml. 50 μl of working internalstandard solution were added to all samples. In addition to the abovestandards and internal controls, the following reagent compositions wereprepared. A 0.1M phosphate buffer, pH 6.0 was prepared by weighing out13.61 g of a reagent grade potassium phosphate monobasic (KH₂ PO₄)available from E-K INDUSTRIES and charging it into a 1.0 litervolumetric flask. The potassium phosphate monobasic was dissolved in 900ml of de-ionized water. The pH was adjusted to 6.0 with 1.0M potassiumhydroxide with stirring. The total volume was brought up to 1.0 literwith de-ionized water. The phosphate buffer reagent was stored at 5° C.and discarded every 30 days. A 1.0M potassium hydroxide solution wasprepared by weighing 5.6 g of potassium hydroxide reagent grade into a100 ml volumetric flask. The potassium hydroxide was dissolved withde-ionized water and brought to volume. The potassium hydroxide standardwas stable at room temperature for three months. A 0.1M hydrochloricacid solution was prepared by adding 150 ml of de-ionized water to a 250ml volumetric flask. 2.1 ml of concentrated hydrocholoric acid ACSreagent grade was added and the solution was further brought to volumewith additional de-ionized water. The hydrochloric acid solution wasstable at room temperature for three months. A methylenechloride:isopropyl alcohol (80:20) solvent solution with 2% ammoniumhydroxide was prepared by adding 80 ml of methylene chloride and 20 mlof isopropanol to a 100 ml reagent bottle. The mix solvents were mixedand 2 ml were removed. Thereafter, 2 ml of ammonium hydroxide reagentgrade were added to return the reagent volume to 100 ml. The 100 ml weretransferred to a capped container and this reagent was prepared daily.

In accordance with this Example procedure, a specialized extraction forcocaine and benzoylecgonine metabolite was performed as follows:

A 1.0 g sample of pre-processed meconium previously testing positive inthe FPIA screening procedure for cocaine was placed in a 16 by 125disposable polypropylene tube. 50 μl of working internal standardscontaining 10 mg/ml of deuterated benzoylecgonine and deuterated cocaineeach were added respectively. 3.0 ml of glacial acetic acid reagentgrade were added to each sample. The acid and spiked sample werehomogenized using an OMNI 5000 tissue homogenizer set at high speed fora period of at least 30 seconds until a substantially uniform homogenatewas obtained. 6.0 ml of acetone and diphenylamine prepared in accordancewith the method of Example 1 were added to each sample. The samples werevortexed and allowed to mix on a sample mixer for a period of about 5minutes. Thereafter, the mixed two-phase samples were centrifuged athigh speed for 5 minutes to resolve the two-phase mixture into a toporganic phase and a lower second phase containing the meconium solids.The top organic phase was carefully transferred to a silanized 10 mlconcentrator cup. The concentrator cup was placed in a sampleconcentrator set at 75° C. over air and vacuum. The volume of theorganic phase in the concentrator cup was reduced to less than 0.5 ml,care being taken to make sure that the organic phase did not bump.Thereafter, 0.5 ml of absolute methanol was added to each concentratedsample and the sample was mixed thoroughly. The mixed sample wastransferred to a clean 16 by 125 ml polypropylene tube and 6.0 ml of the0.1M phosphate buffer pH 6 were added to each tube. The bufferedconcentrates were vortexed to mix thoroughly. Thereafter, the bufferedconcentrates were centrifuged at high speed for 5 minutes and any lipidlayer at the top of the tube was carefully aspirated prior toproceeding. The outlet ends of an appropriate number of BOND ELUTE(CERTIFY) ONE brand extraction columns (VARIAN) were placed into thefemale end of the stock cock valves on the top of the vacuum manifold.An appropriate number of labelled polypropylene 16×125 mm tubes wereemployed as collection vessels and placed under each column in thevacuum manifold. The BOND ELUTE certified columns were conditioned bysequentially passing through each column a 2.0 ml of absolute methanolfollowed by 2.0 ml of the 0.1M phosphate buffer pH 6. The column bedswere not permitted to dry. The stock cock valves were closed as soon asthe liquid phase reached the top of the frit.

Each buffered specimen was filtered through a 30 micron polypropylenefrit as it was poured into the column reservoir. The vacuum was turnedon and adjusted by the controller so that the flow rate through thecolumn was about 2.0 ml per minute (5 to 10 inches of mercury).Thereafter, the column was sequentially rinsed by passing through thecolumn 3.0 ml of de-ionized water, 3.0 ml of 0.1M hydrochloric acidsolution and 9.0 ml of methanol. Receiving tubes were placed in themanifold rack and all needle tips were cleaned with lab tissue beforereplacing the manifold top. The rack was then placed within the manifoldand the target analytes and analyte analogs were eluted by passingthrough each column 2.0 ml of the methylene chloride/isopropanol (80/20,v/v) plus 2% NH₄ OH reagent solution prepared above. The eluate wastransferred to 3.0 ml aluminum concentration vials and the extracts weredried on a hotplate over hot air with care being taken that the eluatephase does not bump. The residues were derivatized by adding 100 ml of95% reagent grade ethanol to the dried residue. After swirling andmixing, the residue was transferred to auto-sampler vials containing 250μl silanized inserts. The reconstituted residues were again taken todryness in a vacuum oven set at 55° C. and 20 to 22 mm of mercury. Theauto-sampler vials were capped securely and 40 μl ofN-methyl-N-trimethylsilyltrifluorilacetamide (Pierce Chemicals) wasadded to each vial and the contents of each vial were vortexed.Thereafter, the vortexed mixture was heated for 15 minutes at 75° C. ina dry bath until derivatization was complete. 2.0 μl of the derivatizedtest solution was injected into the GC for analysis.

The parameters for the GC/MS operation were as follows: The columnconsisted of 5% phenyl-95% methylsilicone, 0.20 mm ID, 0.33 micron filmthickness, 25M length. The GC conditions were set as follows: injectortemperature--250° C.; transfer line temperature--270° C.; starttemperature--100° C.; injection mode--splitless; purge time on--0.5minutes. The instrument protocol was set as in the following programsequence: 100° hold 1 minute, ramp at 20° per minute until 300°, holdfor 1 minute.

The GC/MS acquisition parameter file was setup to determine two groupsof selected ions as follows:

    ______________________________________                                                      Quantitative Ion                                                                           Qualifiers                                         ______________________________________                                        DRUG: Cocaine   303            182     82                                     DRUG: Benzoylecgonine                                                                         361            240     82                                     INTERNAL STANDARDS                                                            D.sub.3 - Cocaine                                                                             306                   185                                     D.sub.3 - Benzoylecgonine                                                                     364                   243                                     ______________________________________                                    

The samples were run through the GC/MS instrument and curves for theselected ions were generated.

The calculations used to determine quantitatively the amount of targetanalyte present in the meconium sample was based on comparing the nativedrug's response against the quantitatively known analogs. This techniqueallowed quantitative calculations based on a ratio of the native drugarea versus the labelled analog area. This ratio is then multiplied bythe amount of labelled analog in the extract in nanograms divided by theextracted amount in grams of sample. The internal standards weredeuterated analogs of the compounds of interest and extractionrecoveries and instrument responses are effectively equal. These twoassumptions eliminate the requirement for recovery determinations sinceany factor affecting the response of the native drug will also affectequally the response of the deuterated analogs. The calculations may berepresented by the following formula: ##EQU1## One criteria for thereporting of data was for the calculated ion ratio to be a certainpercentage of expected ratios. These ratios were calculated using TARGETsoftware on the Hewlett-Packard GC/MS data system. A relative 30% windowwas used for acceptable data so that an ion with an expected ratio of60% would have a 42% to 78% window for an acceptable ion ratio range.

In accordance with this confirmatory procedure, the cocaine reportingrange was determined to be from 5.0 ng/g to 10,000 ng/g for the meconiumsamples.

EXAMPLE 9 GC/MS Confirmatory Procedures For Quantitative Determinationof Amphetamines in Meconium Samples

Generally, in accordance with this method meconium specimens previouslytesting positive by FPIA for amphetamines are extracted under alkalineconditions using liquid/liquid techniques. The samples are thereafterderivatized and analyzed by GC/MS using electron impact and selected ionmonitoring mode.

Prior to extracting a second aliquot sample presumed positive by FPIA,the following reagent solutions and standards and controls wereprepared. A 0.1% methanolic H₂ SO₄ solution was prepared by mixing 100ml of absolute methanol and 100 μl of concentrated sulfuric acid to forma stock reagent solution. The sulfuric acid solution was made freshmonthly. An extract salt/buffer mixture was prepared by mixing 6 partsof sodium chloride and 2 parts of sodium carbonate based on the weightof the overall salt/buffer mixture.

A special mixed organic extraction solvent was prepared by admixingvolumetric amounts of heptane/methylene chloride/ethylenechloride/isopropanol in 50/17/17/16 ratios. A 2.40N aqueous hydrochloricacid solution was prepared by admixing 200 ml of concentratedhydrochloric acid with 800 ml of de-ionized water. A 0.10N aqueoushydrochloric acid solution was prepared by mixing 991.7 ml of de-ionizedwater with 8.3 ml concentrated hydrochloric acid.

A 1.0 mg/ml d-amphetamine standard stock solution was prepared by adding31.8 mg of d-amphetamine hydrochloride to a 25 ml volumetric flask anddiluting to volume with absolute methanol. A 1.0 mg/mm methamphetaminestandard stock solution was prepared by adding 31.0 mg ofmethamphetamine hydrochloride to a 25 ml volumetric flask and dilutingto mark with absolute methanol.

A stock internal standard was obtained from RADIAN including ad-amphetamine-d5 solution containing 100 mg/ml and a methamphetamine-d8standard solution containing 100 mg/ml. Working stockamphetamine/methamphetamine controls at 50 mg/ml concentration wereprepared by adding 5.0 ml of the stock amphetamine and methamphetaminestandards to a 100 ml volumetric flask and diluting to mark withabsolute methanol. A working amphetamine methamphetamine control at 100mg/ml concentration was prepared by adding 5.0 ml each of the stockamphetamine and methamphetamine controls to 100 ml volumetric flask anddiluting to mark with absolute methanol. Finally, a working internalstandard having a concentration of 10.0 mg/ml was prepared bytransferring the contents of the stock d-amphetamine-d5 andmethamphetamine-d8 solutions to a 10.0 ml volumetric flask and dilutingto mark with absolute methanol. The negative control sample employed inthe confirmatory assay comprised in-house drug-free meconium spiked with100 ng/g methamphetamine and amphetamine.

In accordance with the amphetamine confirmatory procedure 0.5 to 1.0 gof presumptive positive meconium sample are placed in 16 by 125 mlpolypropylene tubes. The samples are spiked with 10 μl of the workinginternal standards containing both d-amphetamine d5 andmethamphetamine-d8. Thereafter, 5.0 ml of the 2.4N aqueous HCL solutionwas added to each sample. The acidified samples were homogenized usingthe OMNI 5000 tissue homogenator set at high speed for periods of atleast 30 seconds until substantially uniform homogenates were prepared.The homogenate was centrifuged to define an aqueous acid top layer and asecond pellet layer containing meconium solids. The top aqueous layerwas transferred to clean 16 by 125 silanized glass screw-top tubes.Thereafter, the acidified samples were neutralized by adding 0.8 ml of a12N sodium hydroxide solution followed by vortex mixing. Afterneutralizing, 2.0 g of the extraction/salt/buffer mixture were added toeach tube. Thereafter, 5.0 ml of the mixed organic extraction solventwas added to each tube and the contents of each tube were mixedthoroughly for 5 minutes. Thereafter, the contents of the tube werecentrifuged for 5 minutes and the top organic phase was transferred toclean 16 by 125 ml tubes. After being transferred to clean 16 by 125tubes, the intermediate extract was again acidified by adding 2.0 ml ofthe 0.1N HCL solution to each tube. After mixing for 5 minutes, thesolutions were centrifuged at high speed for 5 minutes and any toporganic phase was aspirated and discarded. Thereafter, 2.0 g of thesalt/buffer mix were added to each tube and 2.5 ml of the mixed organicextraction solvent were added and this mixture was again mixed for 5minutes. Once again, this mixture was centrifuged at high speed for 5minutes and the top organic phase was transferred to 3.0 ml aluminumvials. A drop of the 0.1% sulfuric acid in methanol was added to eachtube to prevent entraining of analyte. Thereafter, the organic sampleswere taken to dryness on a hotplate with forced air. The samples werereconstituted with a 100 μl of ethyl alcohol reagent grade with swirlingto aid dilution. The alcohol solution was transferred to auto-samplervials and again taken to dryness in a vacuum oven set at 55° C. with avacuum of 20 to 22 of mercury. 30.0 μl of isooctane and 10.0 μl of anN-methylbis(heptafluorobutyramide) derivatizing agent were added. Thesealed vials were then placed in heating blocks at 75° C. for 30 minutesto permit derivatization to proceed until substantially complete. Thederivatized samples were cooled to room temperature prior to injectinginto the GC/MS for determination in accordance with the method ofExample 8. For this amphetamines confirmatory procedure the GCconditions were set as follows: Injector temperature--225° C.; transferline temperature--250° C.; start temperature--100° C., hold for 1minute; the injection mode--splitless; and purge time on was 0.5minutes. The program protocol was set at 100°, hold 1 minute, ramp at20°/minute to 260° and hold for 1 minute. The mass monitored ions wereselected as follows:

    ______________________________________                                        Amphetamine    240        118     91                                          Methamphetamine                                                                              254        210    118                                          Amphetamine D5 244        123                                                 Methamphetamine D-8                                                                          261        213                                                 ______________________________________                                    

The concentration of amphetamine and methamphetamine target analytespresent in the meconium samples was determined in ng/g by calculatingthe ratio of the response area of the amphetamine or methamphetaminenative drug area versus the area of the labelled deuterated analogstimes the amount of the labelled drug divided by the extracted mass asset forth in accordance with the formula in Example 8.

In accordance with the new and improved confirmatory procedure thecut-off limit for detecting amphetamine or methamphetamine targetanalytes in a meconium sample is 50 ng/g. It has been determined thatthe limits of linearity for the GC/MS confirmatory assay procedureextends from 50 to 5,000 ng/g Values over 5,000 ng/g are simply reportedas >5,000 ng/g Values less than 50 ng/g are reported as a negativefinding for amphetamine or methamphetamine analyte.

EXAMPLE 10 GC/MS Confirmatory Procedure For Quantitative Determinationof Opiate Analytes in Meconium Samples

In accordance with this general procedure, meconium specimens previouslytesting to be as presumptive positive by FPIA in accordance with themethod of Example 4 are extracted under alkaline conditions usingliquid/liquid extraction techniques. The samples are derivatized andthen analyzed by GC/MS using electron impact and selected ion monitoringmode. The GC/MS procedural protocols generally followed the proceduresoutlined in Examples 8 and 9 identified above. The standard reagentsolutions and calibration standards prepared for this assay were asfollows: An extraction salt buffer mixture was prepared by mixing 6parts of sodium chloride, 1 part of sodium bicarbonate and 1 part ofsodium carbonate based on the total weight of the salt/buffer mixture. A2.4N aqueous HCL solution was prepared by mixing 207 ml of concentratedreagent grade HCL with 793 ml of de-ionized water.

Calibration standards and internal controls were prepared by taking 100ng each of morphine and codeine spiked with 500 ng each of deuteratedanalogs. The standards are derivatized identically to authentic meconiumextracts and analyzed. The peak area ratio is placed into the targetcalibration files. The deuterated analyte analogs used as internalstandards selected were morphine-D₃ and codeine-D₃ in concentrations of0.1 mg/ml in methanol. Working internal standards for morphine-D₃ andcodeine-D₃ were prepared to contain 10 mg/ml of the internal standards.50.0 μl of the internal standards were added to all tubes. Thedeuterated morphine and codeine reagents were supplied by RADIANCORPORATION, Austin, Tex. Negative internal controls were prepared fromin-house drug-free meconium spiked at 100 ng/g with 100 μl of the lowmeconium spiking control samples.

In accordance with the new and improved opiate assay confirmatory assay,an extraction was performed by placing 0.5 to 1.0 g of pre-processedmeconium in a 16 by 125 polypropylene tube. 50.0 μl of working internalstandards containing both codeine-D₃ and morphine-D₃ were added to eachtube. Thereafter, 5.0 ml of 2.4N aqueous hydrochloric acid solution wereadded to each tube and specimen. The contents of each tube werehomogenized using the OMNI 5000 tissue homogenator for a period of atleast about 30 seconds to achieve a smooth, uniform homogenate. Theacidified homogenized samples were then centrifuged at high speed for 5minutes. The top aqueous layer was transferred to a new 16×125 silanizedglass screw-top tube and 5 ml of methytertbutylether were added andmixed for a period of about 5 minutes. The two-phase mixture waspermitted to separate and the top organic layer was carefully aspiratedand discarded. The aqueous acid layer was then neutralized with 0.8 mlof 12N sodium hydroxide and vortexed. Thereafter, 7.5 ml ofmethyl-t-butyl ether were added to each tube and 2.0 g of thesalt/buffer mixture were added to each tube. The neutralized bufferedmixtures were mixed for 5 minutes, centrifuged at high speed for 5minutes and the top organic phase was transferred to 10.0 ml glassconcentration vials. The contents of the glass concentration vials wereevaporated to dryness in an Alltech sample evaporator under air andvacuum. Target analyte and analog values were reconstituted in 100 μl ofethanol with swirling to aid dilution. The ethanolic solutions weretransferred to auto-sampler vials and again evaporated to dryness in avacuum of and set at 75° C. and a vacuum of 20 to 22 mm of mercury.After being tightly capped, 40.0 μl ofN-methyl-N-trimethylsilyltrifluoroacetamide derivatizing agent fromPierce Chemicals was added. The vials were placed in heating block at75° C. for 10 minutes to permit the derivatization reaction to proceedto completion. The derivatized samples were then permitted to cool toroom temperature before injecting in the GC/MS instrument.

The GC/MS injection procedure was substantially in accordance with themethod outlined in Examples 8 and 9. The GC conditions were set so thatthe injector temperature was 250° C., the transfer line temperature was270° C., the start temperature 100° C. hold for 1 minute, the injectionmode was splitless and the purged time on was 0.5 minutes. The firstprogram was set to heat to 100°, hold for 1 minute, ramp at 20° perminute to 300° and hold for 1 minute. The following ion peak qualifyingparameters were employed:

    ______________________________________                                        DRUG           ION    DWELL TIME                                              ______________________________________                                        GROUP 1                                                                       Codeine D.sub.3                                                                              346    50                                                      Codeine D.sub.3                                                                              374    50                                                      Codeine        343    50                                                      Codeine        234    50                                                      Codeine        371    50                                                      GROUP 2                                                                       Morphine-D.sub.3                                                                             417    50                                                      Morphine D.sub.3                                                                             432    50                                                      Morphine       401    50                                                      Morphine       414    50                                                      Morphine       429    50                                                      ______________________________________                                    

The ion parameters are set to give the internal standard quantificationion area count greater than 50,000 and allow for reproducibleacquisition of 3 ions for the native drug and 2 ions for its deuteratedanalog. The concentration of opiates present was calculated by comparingthe ratio of the response area for the native drug versus the responsearea of the labelled analog multiplied by the amount of labelled drug innanograms divided by the extracted mass in grams. Once again because ofthe use of internal standards and controls the response factor is 1.

The GC/MS confirmatory procedure for opiates has a lower cut-offdetection concentration limit of 25 ng/g. The limits of linearity forthe procedure are from 25 ng to 1,000 ng/g. Values greater than 1,000ng/g are reported as >1,000 ng/g and values less than 25 ng/g arereported as negative for opiate analytes.

EXAMPLE 11 GC/MS Confirmation Procedure For Quantitatively DeterminingTetrahydrocannabinol Analyte in Meconium Samples

A GC/MS extraction technique and determination was performed on meconiumsamples previously testing positive by the preliminary FPIA screeningmethod of Example 5. Generally, in accordance with this GC/MSconfirmatory procedure a presumptive positive sample of meconium isspiked with deuterated THC-COOH analog followed by homogenization,alkaline hydrolysis, acidification, liquid/liquid extraction followed byderivatization. More particularly, the following reagent solutions,calibrators and controls were prepared prior to extraction.

The calibration standards employed in the assay were for 100 ng of11-nor-Delta-9-tetrahydrocannabinol-9-carboxylic acid spiked with 100 ngof a deuterated analog. The deuterated analog, THC-COOH-D₃ is used asthe internal standard as a solution of 0.1 mg/ml in methanol. Theworking internal standard for THC-COOH-D₃ was 2.0 mg/ml and 50 μl wereadded to all tubes.

An organic extraction solvent mixture of N-hexane/ethyl acetate(90/10,v/v) was prepared by adding 100 ml of ethyl acetate to 900 ml ofN-hexane in a reagent bottle. An 11.8N potassium hydroxide solution wasprepared by dissolving 66.2 g of potassium hydroxide in 75 ml ofde-ionized water in a 150 ml beaker. Once dissolved the solution waspoured into a 100 ml volumetric flask and filled with de-ionized waterto the volume mark.

An in-house 100 ng calibrator or equivalent was analyzed with each runand calibration was performed on the H-P data system using TARGETsoftware. In addition, in-house pooled meconium positive samples forcannabinoid known to contain 58 ng/g of THC-COOH are added in a blankdrug-free meconium sample are also run with each batch as qualitycontrols.

In accordance with this new and improved confirmatory method forquantifying THC metabolite, a 0.5 to 1.0 g sample of pre-processedmeconium testing as presumptively positive by FPIA was placed in 16 by125 mm polypropylene sample tubes. 50 μl of a working internal standardare added, 3.0 ml of methanol are added and the samples are homogenizedusing an OMNI 5000 tissue homogenizer set at high speed for a period ofabout at least 30 seconds until a homogenate is formed. The homogenizedsample is permitted to stand for at least 5 minutes. Thereafter, 0.5 mlof an 11.8N potassium hydroxide solution is added to each tube and thecontents are vortexed. After thorough mixing, the contents arecentrifuged at high speed for 5 minutes and the top organic phase istransferred to a clean 16 by 125 silanized glass tube. 3.0 ml ofde-ionized water and 6.0 ml of the mixed ethyl acetate/N-hexaneextraction solvent are added to each tube. The contents are vortexed andthen permitted to incubate on a sample mixer for a period of about 15minutes. The top organic phase is aspirated and discarded and 0.8 ml ofconcentrated hydrochloric acid are added to each tube and the contentsare vortexed. 8.0 ml of the mixed extraction solvent are added to thetube and the sample is again mixed for a period of about 5 minutes.After centrifuging at high speed for 5 minutes, the top organic phase istransferred to silanized concentration cups and placed in a sampleconcentrator set at 75° C. and dried over air and vacuum. The driedresidues are reconstituted with 100 μl of ethanol and the ethanolsolution is transferred to an auto-sampler vial containing 250 μlsilanized liners. Again the sample is taken to dryness in a vacuum ovenset at 70° to 85° C. at a vacuum of 20 to 25 inches of mercury withoutpermitting the solvents to bump. Derivatization was accomplished bycapping and crimping each auto-sampler vial and thereafter adding 25 μlof N-methyl-N-trimethylsilyltrifluoroacetamide derivatizing agent toeach vial. The contents of each vial are permitted to react andderivatize for a period of about at least 15 minutes at 75° C.Thereafter, the derivatized sample is permitted to cool prior to beinginjected into the GC/MS instrument. 2.0 μl of the derivatized sample areinjected into the GC/MS system. The selected ions monitored for THC werenon-deuterated 371.30, 473.30, 488.30 and for the deuterated THC ions at374.30 and 491.30.

The samples and calibrators were run through the GC/MS instrument andthe concentration of THC carboxylic acids were determined by determiningthe ratio of the native drug area versus the labelled analog area andthereafter multiplying that ratio by the amount of labelled analytepresent in the extract divided by extract mass to yield theconcentration of marijuana metabolite present in the meconium sample inng/g of sample tested. The reporting range for this confirmatory assayhas been determined to be from about 10 to 200 ng/g. Samples quantifyingat less than 10.0 ng/g are reported as negative. Those quantifying asgreater than 200 ng/g are reported as greater than 200 ng/g. For theabove procedures the GC conditions were set at 250° C. injectortemperature, the transfer line temperature was 270° C., the startingtemperature was 100° C., holding for 1 minute, the injection mode wasset at splitless and the purge time on was 0.5 minutes. The program usedwas 100°, hold 1 minute, ramp at 20° per minute to 320°.

EXAMPLE 12 GC/MS Confirmatory Procedure For Quantitative Determinationof Phencyclidine Analyte in Meconium Samples

In accordance with this example, a quantitative determination ofphencyclidine concentration is made by performing a solid phaseextraction of homogenized and protein denatured meconium samplepreviously testing as presumptively positive by FPIA assay forphencyclidine. The phencyclidine concentration is confirmed and thelevel determined by monitoring its major ions and comparing them withspiked internal standards. Prior to an extraction procedure thefollowing calibration standards, reagents and internal controls wereprepared. As calibration standards, 25 ng of phencyclidine spiked with50 ng of the deuterated analog PCP-D₅ were prepared. Phencyclidine-D₅ isused as the internal standard at 0.1 mg/ml in HPLC grade methanol. Theworking internal standard for PCP-D₅ is 10.0 mg/ml and 25 μl were addedto all tubes. The phencyclidine-D₅ deuterated drug analog is supplied byRADIAN CORPORATION, Austin, Tex. The following reagents were prepared: A0.1M phosphate buffer having a pH of 6.0 was prepared by adding 13.61 gof potassium phosphate monobasic into a 1.0 liter volumetric flask. 900ml of de-ionized water were added to dissolve the potassium phosphatemonobasic and the pH was adjusted to 6.0 with 1M potassium hydroxidewhile stirring. Thereafter, the total volume was diluted to mark withde-ionized water. The solution was stored at 5° C. and discarded every30 days. A 1M potassium hydroxide solution was prepared by weighing 5.6g of potassium hydroxide and adding it to a clean 100 ml volumetricflask. The potassium hydroxide was dissolved with de-ionized water anddiluted with de-ionized water to the volume mark. This reagent solutionwas stable at room temperature for three months. A 1.0M acetic acidsolution was prepared by adding 15 ml of de-ionized water to a 100 mlvolumetric flask. 5.75 ml of glacial acetic acid were added to the flaskand the flask was diluted to volume with additional de-ionized water.This reagent solution was stable at room temperature for 2 months. Anethyl acetate with 2% ammonium hydroxide reagent solution was preparedby adding 98 ml of ethyl acetate to 100 ml reagent bottle and thereafteradding 2.0 ml of ammonium hydroxide. The ethyl acetate 2% ammoniumhydroxide solution was prepared fresh daily. A 1.67 mg/literdiphenylamine in acetone reagent solution was prepared by adding 6.67 mgof diphenylamine into a new 4 liter bottle of reagent grade acetone. Thecontents were shaken to mix thoroughly and were stable at roomtemperature for 12 months. Finally a 0.1% sulfuric acid in methanolsolution was prepared by adding 100 μl of concentrated sulfuric acid to100 ml of methanol and storing the solution in a capped container.

Controls for the assay were prepared by taking known in-house drug-freemeconium samples and spiking them at a loading level of 50 ng/g. Also anegative drug-free meconium sample was run as a calibrated control.

In accordance with the new and improved confirmatory extraction method a0.5 to 1.0 g sample of meconium previously tested presumptively positivefor PCP content was placed in a 16 by 125 polypropylene tube. 25 μl ofthe deuterated PCP analog PCP-D₅ are added to each specimen tube. 3.0 mlof glacial acetic acid are added to each specimen and the acidifiedtissue sample was homogenized using a tissue homogenizer set at highspeed for at least about 30 seconds. Thereafter, 6.0 ml of theacetone/diphenylamine reagent is added to each tube. The contents of thetubes are vortexed and then allowed to mix for 5 minutes. The organicphase is separated from a second phase containing meconium solids bycentrifugation at high speed for a period of at least about 5 minutes.The top organic phase was transferred to a 10 ml concentration cup. Adrop of 0.1% H₂ SO₄ in MeOH was added to each tube to preventinadvertent loss of analyte. Thereafter, the concentration cups wereplaced in a sample concentrator at 75° C. over air and vacuum and thevolume of each sample was reduced to less than about 0.5 ml, care beingtaken that the samples do not bump during drying. Thereafter, 0,5 ml ofmethanol were added to each cup and the samples were mixed thoroughlyand transferred to clean 16 by 125 polypropylene tubes. 6.0 ml of 0.1Mphosphate buffered pH 6.0 were added to each tube and the solutions werevortexed thoroughly. Thereafter, the solutions were centrifuged at highspeed for 5 minutes and any lipid top layer is very carefully aspiratedand discarded before proceeding. An appropriate of BOND ELUTE (CERTIFY)ONE solid phase extraction columns are attached to the female ends ofstock cock valves mounted on the top of the vacuum manifold. Anappropriate number of labelled polypropylene tubes as collection vesselswere placed under each column in the vacuum manifold. Each of thecolumns was conditioned by sequentially passing through each column 2.0ml of methanol and 2.0 ml of the 0.1M phosphate buffer having a pH of6.0. The columns were not permitted to go to dryness. Each of theprepared buffered specimens were passed into column reservoirs and thevacuum was adjusted so that the flow rate through the column was set atabout 2.0 ml/minute (5-10 inches of mercury). After the sample waspassed through the column, the column was rinsed sequentially by passingthrough 1.0 ml of a 1.0M acetic acid solution and dried under fullvacuum for 5 minutes. Thereafter, 6.0 ml of methanol are flowed throughthe column and the column is dried under full vacuum for 2 minutes.Receiving tubes were placed in the manifold rack and needle tips werewiped with lab tissue and the manifold top was replaced. Phencyclidinetarget analytes and analyte analogs were eluted from the column bypassing through each column 2.0 ml of the ethyl acetate containing 2%ammonium hydroxide solution. The eluate is transferred to a 3.0 mlaluminum concentration vial and 1 drop of 0.1% sulfuric acid andmethanol is added to each tube. The extracts were then dried on ahotplate over hot air making sure the extracts do not bump. The driedresidues were reconstituted with 50 μl of a mixed isooctane/isopropanol80/20 V/V mixed solvent. The reconstituted residue is transferred toauto-sampler vials containing silanized 250 μl limited volume inserts.The caps are placed and crimped on each vial and the samples areinjected into GC/MS.

The gas chromatograph settings were set at initial temperature of 80°C., the transfer line temperature was set at 250° C., the starttemperature was set at 80° C., hold for 1 minute. Injection mode wassplitless, the purge time on was 0.5 minutes. The program was set to runat 80°, hold for 1 minute, ramp at 22.5° C./minute to 250° C.

The ions monitored were for phencyclidine, quantitative ion--200 andqualifiers at 243/200 and 242/200. The phencyclidine internal standardcontrol quantitative ion was 205 and single qualifier at 248/205 wasused. The samples and negative controls and calibrators were run throughthe GC/MS instrument and the amount of PCP present in the meconiumsample was quantitatively determined. The PCP reporting range for thisassay has been determined to be from 25 ng to about 200 ng/g ofmeconium. Samples quantifying at as having less than 25 ng/g arereported as negative. Those samples quantifying at greater than 200 ng/gare simply reported as greater than 200 ng/g.

I claim:
 1. A method for preparing a concentrated neonatal meconiumextract containing substantially all of at least one target analytepresent, if any, in a sample of infant meconium suspected of containingsaid at least one target analyte, said at least one target analyte isselected from the group consisting of amphetamine, opiate, cocainemetabolite and cannabinoids, said concentrated meconium extract beingspecifically intended for use in subsequent qualitative and quantitativedeterminations of maternal use or exposure to said at least one targetanalyte during pregnancy and fetal exposure to said at least one targetanalyte, said method comprising:providing a test sample of newbornmeconium suspected of containing said at least one target analyte;adding a substantially non-aqueous, volatile organic acid to said sampleto form a first mixture; agitating the first mixture for a timesufficient to extract substantially all of said at least one targetanalyte from the meconium sample, without diluting the sample withwater, until a substantially uniform first liquid homogenate isobtained; adding a volatile organic solvent to said first liquidhomogenate to form a two-phase mixture including an organic phase and asecond phase; thoroughly mixing the two-phase mixture for a timesufficient to permit substantially all of said extracted at least onetarget analyte to be extracted and collected into said organic phase;and thereafter, separating the organic phase from the second phase,wherein said organic phase is concentrated to provide said concentratedextract.
 2. A method as defined in claim 1, wherein said sample ofinfant meconium includes a semi-solid meconium matrix portion.
 3. Amethod as defined in claim 2, wherein said at least one target analyteis bound in said meconium matrix.
 4. A method as defined in claim 3,wherein said at least one target analyte is protein-bound in saidmeconium matrix.
 5. A method as defined in claim 1, wherein said atleast one target analyte comprises a detectable chemical compound or ametabolite thereof capable of becoming protein-bound in a physiologicalsystem, after being introduced into the physiological system byingestion, insufflation, inhalation, subcutaneous injection, venousinjection, placental transfer, absorption or adsorption.
 6. A method asdefined in claim 1, wherein the step of providing a sample of newbornmeconium comprises collecting and pooling meconium produced by an infantnewborn from birth until just prior to the appearance of a transitionalmilk stool; andvigorously mixing the pooled and collected meconium untila substantially uniform, non-striated, smooth semi-solid meconiumproduct having a putty-like consistency is obtained and selecting saidtest sample from a portion of said mixed semi-solid meconium product. 7.A method as defined in claim 6, wherein the step of agitating the firstmixture includes homogenizing the first mixture by subjecting the firstmixture to high shear blade processing to finely subdivide the meconiumcomponent.
 8. A method as defined in claim 7, wherein said homogenizingstep is performed in a tissue homogenizer and the pooled meconium ishomogenized for a time sufficient to provide a substantially uniformhomogenate.
 9. A method as defined in claim 8, wherein said uniformmeconium homogenate is characterized by having an average particle sizeof less than about 10.0 microns.
 10. A method as defined in claim 8,wherein said meconium is homogenized until a substantially uniformhomogenate having an average particle size of from about 2.0 to about5.0 microns is obtained.
 11. A method as defined in claim 6, whereinsaid meconium is collected and pooled by providing a plurality of lineddiapers for use with each suspect infant over the first days of saidinfant's life, each lined diaper including a top liner layer positionedto be contacted by the meconium produced by said infant, said linerbeing selected so that said meconium does not adhere to the liner orbecome absorbed by it, successively diapering said infant newborn with asaid lined diaper until a first transitional milk stool is observed;transferring meconium produced, if any, from each said liner to aclosable storage container and refrigerating said collected and pooledmeconium in said closable storage container until meconium collectionfor said infant is substantially complete.
 12. A method as defined inclaim 11, wherein said diaper liner comprises polyethylene.
 13. A methodas defined in claim 1, wherein each of the steps are performed insilanized glassware.
 14. A method as defined in claim 1, wherein saidsubstantially non-aqueous volatile organic acid is selected fromvolatile organic acids which do not react with said at least one targetanalyte, which exhibit rapid evaporation rates at 25° to 100° C. andwhich do not liberate toxic gases or vapors upon evaporation.
 15. Amethod as defined in claim 14, wherein said volatile organic acid isselected from glacial acetic acid, propionic acid, or butyric acid. 16.A method as defined in claim 1, wherein a glass anti-binding agent isadded with said volatile organic solvent.
 17. A method as defined inclaim 16, wherein said glass anti-blocking agent comprisesdiphenylamine.
 18. A method as defined in claim 1, wherein said volatileorganic solvent is selected from the group consisting essentially ofacetone, acetonitrile and dialkyl ethers.
 19. A method as defined inclaim 1, wherein said volatile organic solvent comprises a polar organicsolvent.
 20. A method as defined in claim 1, wherein said volatileorganic solvent comprises acetone.
 21. A method as defined in claim 1,further comprising reducing the volume of the concentrated meconiumextract by evaporating a portion of volatile components thereof andthereafter reconstituting a part of said evaporated volume with aphosphate buffer.
 22. A method as defined in claim 21, wherein thereconstituted, buffered extract is centrifuged to provide an upperorganic layer containing lipids, fatty acids and triglycerides and alower buffer layer containing target analytes and said buffer layer isseparated to provide a clarified concentrated meconium extract forfurther testing.
 23. A method as defined in claim 1, wherein saidseparating step is performed by centrifuging the thoroughly-mixedtwo-phase mixture to separate the organic phase from said second phase.24. A concentrated meconium extract for use as a starting patient samplematerial in further drug abuse testing comprising the product made bythe method of claim
 1. 25. A concentrated meconium extract for use as astarting patient sample material in further drug abuse testingcomprising the clarified, buffered product made by the method of claim22.
 26. A method for making a qualitative determination of maternal useof or exposure to at least one target analyte during pregnancy andrelated exposure of a subject infant to said at least one target analyteas a fetus, said at least one target analyte is selected from the groupconsisting of amphetamine, opiate, cocaine metabolite and cannabinoids,a qualitative determination of the presence or absence of said at leastone target analyte is made in a sample of neonatal meconium obtainedfrom said subject infant, said method comprising;providing a sample ofneonatal meconium obtained from a newborn infant suspected of beingexposed to said at least one target analyte in utero; contacting saidsample with a substantially non-aqueous, volatile organic acid to form afirst mixture said first mixture for a time sufficient to extractsubstantially all of said at least one target analyte from said meconiumsample until a substantially uniform liquid dispersion is obtained;adding a substantially non-aqueous volatile organic solvent to saidliquid dispersion to form a two-phase mixture including an organic phaseand a second phase; thoroughly mixing said two-phase mixture for a timesufficient to permit substantially all of said extracted at least onetarget analyte, if present, to be extracted and collected in saidorganic phase; separating said organic phase from said second phase toform a substantially non-aqueous concentrated meconium extract fortesting; and thereafter, analyzing said extract for the presence of saidat least one target analyte.
 27. A method as defined in claim 26,wherein said sample of neonatal meconium comprises an aliquot portiontaken from a prepared meconium specimen obtained by collecting andpooling a plurality of meconium bowel movements from said subject infantfrom birth until just prior to a first transitional milk stool;thoroughly mixing said collected and pooled meconium until a preparedmeconium specimen comprising a substantially uniform, non-striated,smooth semi-solid meconium product having a paste-like consistency isobtained.
 28. A method as defined in claim 26, wherein said step ofagitating said first mixture is performed by homogenizing the organicacid and meconium under bladed high shear mixing conditions until asubstantially uniform liquid homogenate is obtained.
 29. A method asdefined in claim 26, wherein said volatile organic acid comprisesglacial acetic acid.
 30. A method as defined in claim 26, wherein saidvolatile organic solvent comprises acetone.
 31. A method as defined inclaim 26, wherein the separating step includes the steps of centrifugingthe two-phase mixture to provide a clearly defined top organic phaselayer and selectively isolating said top organic phase from said lowersecond phase to form said concentrated meconium extract for testing. 32.A method as defined in claim 31, further comprising the steps ofconcentrating the isolated organic phase by evaporating volatilecomponents until the volume of the organic phase is reduced to less thanabout one-half its original isolated volume and thereafter,reconstituting the organic phase to less than or equal to its originalisolated volume by adding an effective amount of a buffer solution toprovide a buffered extract.
 33. A method as defined in claim 32, furthercomprising treating the isolated organic phase with a minor effectiveamount of a sulfating agent prior to said concentrating step to convertvolatile target analytes present in said organic phase to a non-volatilesulfate form to thereby prevent loss of said target analytes bypreventing them from becoming entrained and co-evaporating withevaporating volatile components.
 34. A method as defined in claim 32,further comprising the step of de-lipidizing the buffered extract priorto said analyzing step.
 35. A method as defined in claim 34, whereinsaid de-lipidizing step is performed by centrifuging the bufferedextract to define a top lipid layer and lower buffered extract layer andthereafter removing said top lipid layer to remove fats, lipids, oilsand fatty precursor materials from said buffered extract to form aclarified buffered extract for analysis.
 36. A method as defined inclaim 26, wherein in said analyzing step said concentrated meconiumextract is subjected to at least one immunoassay specific for said atleast one target analyte to determine whether the original neonatalmeconium sample contained any of said at least one target analyte,thereby providing qualitative evidence, of fetal exposure to said targetanalyte and maternal use of or exposure to said target analyte duringpregnancy.
 37. A method as defined in claim 36, wherein said immunoassayspecific for said at least one target analyte comprises an homogeneousimmunoassay.
 38. A method as defined in claim 37, wherein saidhomogeneous immunoassay comprises a fluorescence polarizationimmunoassay (FPIA) method.
 39. A method as defined in claim 36 whereinsaid concentrated meconium extract is subjected to a plurality ofdifferent immunoassays, each being specific for one or more differenttarget analytes.
 40. A method as defined in claim 38, wherein said FPIAimmunoassay is specific for Cannabinoid delta⁹ -tetrahydrocannabinol and11-nor-delta⁹ -THC-9-carboxylic acid target analytes.
 41. A method asdefined in claim 38, wherein said FPIA immunoassay is specific ford,l-amphetamine, d-amphetamine and methamphetamine target analytes. 42.A method as defined in claim 38 wherein said FPIA immunoassay isspecific for benzoylecgonine target analytes.
 43. A method as defined inclaim 38 wherein said FPIA immunoassay is specific for morphine andcodeine target analytes.
 44. A method as defined in claim 38 wherein anypositive immunoassay test result for a target analyte is independentlyquantitatively confirmed by performing a gas chromatographic/massspectroscopic (GC/MS) analysis on a test sample fluid prepared from theneonatal meconium sample obtained from the subject infant.
 45. A methodfor making a forensically acceptable determination of maternal use of orexposure to at least one target analyte during pregnancy and relatedexposure of a subject infant to said at least one target substance as afetus in utero, said at least one target analyte is selected from thegroup consisting of amphetamine, cocaine metabolite, opiate andcannabinoids, said method comprising:(a) providing a first aliquotportion of a time-averaged pooled, pre-mixed meconium tissue sample forthe subject infant; (b) contacting said time-averaged tissue sample witha substantially non-aqueous volatile organic acid to form a firstmixture and agitating said first mixture for a time sufficient toextract substantially all of said at least one target analyte from thetissue sample and until a substantially uniform liquid dispersion isobtained; (c) adding a substantially non-aqueous volatile organicsolvent to said liquid dispersion to form a two-phase mixture includingan organic phase and a second phase; (d) thoroughly mixing saidtwo-phase mixture for a time sufficient to permit substantially all ofsaid extracted at least one target analyte, if present, to be extractedand collected in said organic phase; (e) separating said organic phasefrom said second phase to provide a substantially non-aqueousconcentrated cocktail extract; (f) analyzing at least one aliquotportion of said cocktail extract in a preliminary screening immunoassayspecific for said at least one target analyte to determine whether ornot the at least one target analyte is present in said cocktail extract;and (g) if a positive result, indicating the presence of said at leastone target analyte in said cocktail extract is obtained in step (f),then and thereafter; (h) providing a second aliquot portion of saidtime-averaged meconium tissue sample for the subject infant as in step(a), said second aliquot portion having a known weight; (i) spiking thesecond aliquot portion with a known amount of at least one internalcontrol, said internal control being a labelled analog for saidpositively detected target analyte; (j) treating said spiked secondaliquot portion to release bound target analyte and said added analogsinto a distinct separatable phase; (k) separating said separatable phaseto provide a second extract containing said released target analyte andadded analogs; (l) analyzing said second extract by GC/MS methods toobtain an identifiable response curve associated with said targetanalyte and said analog; and (m) thereafter, quantitatively determiningthe amount of target analyte present in said second aliquot portion interms of nanograms of target analyte per gram of meconium tested bymultiplying the ratio of a response area of target analyte to a responsearea of the known analog times the amount of analog added divided by theextraction mass tested.
 46. A method for making aforensically-acceptable quantitative determination of the concentrationof amphetamine and methamphetamine target analytes present in a meconiumsample, said method comprising:collecting and pooling quantities ofnewborn meconium from a subject infant during the postpartum period;thoroughly mixing the pooled meconium until a substantially uniform,non-striated smooth pre-processed meconium product is obtained;providing a test specimen comprising an aliquot portion of saidpre-processed meconium product having a known weight; spiking said testspecimen with internal controls by adding known amounts of deuterateddrug analogs d-amphetamine-D₅ and methamphetamine-D₈ to said testspecimen; adding an amount of a concentrated aqueous mineral acidsufficient to liberate said amphetamine target analytes and said spikedanalyte analogs into an aqueous acid phase; separating said aqueous acidphase from a second phase containing meconium solids to form a firstacidic extract; subjecting said first acidic extract to additionalsolvent extraction steps to selectively further separate targetamphetamine analytes and analogs present in said first acidic extractfrom other undesired components such that said separated targetamphetamine analytes and analogs end up in organic soluble form in aconcentrated organic solvent solution; derivatizing said target analytesand analyte analogs present in said concentrated organic solventsolution to a derivatized form appropriate for gas chromatography massspectrometric analysis; performing a GC/mass spectral analysis on saidconcentrated derivatized sample solution; and analyzing the results toquantitatively determine the amount of each said target analyte presentin the meconium test specimen in terms of nanograms of amphetamine ormethamphetamine analyte per gram of test specimen meconium bydetermining and multiplying the ratio of a response area of targetanalyte to a response area of the known analyte analog times the knownquantity of the analyte analog added, divided by the mass of meconiumtest specimen.
 47. A method for making a forensically-acceptablequantitative determination of the concentration of benzoylecgoninetarget analyte present in a meconium sample, said methodcomprising:collecting and pooling quantities of newborn meconium from asubject infant during the postpartum period; thoroughly mixing thepooled meconium until a substantially uniform, non-striated smoothpre-processed meconium product is obtained; providing a test specimencomprising an aliquot portion of said pre-processed meconium producthaving a known weight; spiking said test specimen with internal controlsby adding known amounts of deuterated drug analogs andbenzoylecgonine-D₃ to said test specimen; adding a substantiallynon-aqueous, volatile organic acid to said test specimen to form a firstmixture; agitating the first mixture for a time sufficient to releasesubstantially all of said benzoylecgonine target analyte from themeconium sample, without diluting the sample with water, until asubstantially uniform first liquid homogenate is obtained; adding avolatile organic solvent to said first liquid homogenate to form atwo-phase mixture including an organic phase and a second phase;thoroughly mixing the two-phase mixture for a time sufficient to permitsubstantially all of said released benzoylecgonine target analyte andthe analog to be extracted and collected into said organic phase;separating the organic phase from the second phase to provide a firstorganic extract; subjecting said first organic extract to additionalsolvent extraction steps to selectively further separate target andbenzoylecgonine analyte and analog present in said first organic extractfrom other undesired components such that said separated target andbenzoylecgonine analyte and analog end up in organic soluble form in aconcentrated organic solvent solution; derivatizing said target analyteand analyte analog present in said concentrated organic solvent solutionto a derivatized form appropriate for gas chromatography/massspectrometric analysis; performing a GC/mass spectral analysis on saidconcentrated derivatized sample solution; and analyzing the results toquantitatively determine the amount of each said target analyte presentin the meconium test specimen in terms of nanograms of benzoylecgonineanalyte per gram of test specimen meconium by determining andmultiplying the ratio of a response area of target analyte to a responsearea of the known analyte analog times the known quantity of the analyteanalog added, divided by the mass of meconium test specimen.
 48. Amethod for making a forensically-acceptable quantitative determinationof the concentration of opiate morphine and codeine target analytespresent in a meconium sample, said method comprising:collecting andpooling quantities of newborn meconium from a subject infant during thepostpartum period; thoroughly mixing the pooled meconium until asubstantially uniform, non-striated smooth pre-processed meconiumproduct is obtained; providing a test specimen comprising an aliquotportion of said pre-processed meconium product having a known weight;spiking said test specimen with internal controls by adding knownamounts of deuterated drug analogs codeine-D₃ and morphine-D₃ to saidtest specimen; adding an amount of a concentrated aqueous mineral acidsufficient to liberate said opiate target analytes and said spikedanalyte analogs into an aqueous acid phase; separating said aqueous acidphase from a second phase containing meconium solids to form a firstacidic extract; subjecting said first acidic extract to additionalsolvent extraction steps to selectively further separate target opiateanalytes and analogs present in said first acidic extract from otherundesired components such that said separated target opiate analytes andanalogs end up in organic soluble form in a concentrated organic solventsolution; derivatizing said target analytes and analyte analogs presentin said concentrated organic solvent solution to a derivatized formappropriate for gas chromatography/mass spectrometric (GC/MS) analysis;performing a GC/MS analysis on said concentrated derivatized samplesolution; and analyzing the results to quantitatively determine theamount of each said target analyte present in the meconium test specimenin terms of nanograms of codeine and morphine analyte per gram of testspecimen meconium by determining and multiplying the ratio of a responsearea of that target analyte to a response area of the known analyteanalog times the known quantity of the analyte analog added, divided bythe mass of meconium test specimen.
 49. A method for making aforensically-acceptable quantitative determination of the concentrationof 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH)target analyte present in a meconium sample, said methodcomprising:collecting and pooling quantities of newborn meconium from asubject infant during the postpartum period; thoroughly mixing thepooled meconium until a substantially uniform, non-striated smoothpre-processed meconium product is obtained; providing a test specimencomprising an aliquot portion of said pre-processed meconium producthaving a known weight; spiking said test specimen with internal controlsby adding known amounts of deuterated drug analog THC-COOH-D₃ to saidtest specimen; adding an amount of an alkanol solvent sufficient toliberate said target analyte and said spiked analyte analog into saidalkanol phase; separating said alkanol phase from a second phasecontaining meconium solids to form a first alkanol extract; subjectingsaid first alkanol extract to additional solvent extraction steps toselectively further separate target THC-COOH analytes and analogspresent in said first alkanol extract from other undesired componentssuch that said separated target analytes and analogs end up in organicsoluble form in a concentrated organic solvent solution; derivatizingsaid target analytes and analyte analogs present in said concentratedorganic solvent solution to a derivatized form appropriate for gaschromatography/mass spectrometric analysis; performing a GC/MS analysison said concentrated derivatized sample solution; and analyzing theresults to quantitatively determine the amount of target analyte presentin the meconium test specimen in terms of nanograms of THC-COOH analyteper gram of test specimen meconium by determining and multiplying theratio of a response area of target analyte to a response area of theknown analyte analog times the known quantity of the analyte analogadded, divided by the mass of meconium test specimen.
 50. A method forpreparing a concentrated neonatal meconium extract containingsubstantially all of at least one target analyte present, if any, in asample of infant meconium suspected of containing said at least onetarget analyte, said at least one target analyte is selected from thegroup consisting of amphetamine, cocaine metabolite, opiate andcannabinoids said concentrated meconium extract being specificallyintended for use in subsequent qualitative and quantitativedeterminations of maternal use or exposure to said at least one targetanalyte during pregnancy and fetal exposure to said at least one targetanalyte in utero, said method comprising:providing a test sample ofnewborn meconium suspected of containing said at least one targetanalyte, adding a substantially non-aqueous, volatile organic acid tosaid sample to form a first mixture; agitating the first mixture for atime sufficient to extract substantially all of said at least one targetanalyte from the meconium sample, without the diluting the sample withwater, until a substantially uniform first liquid homogenate isobtained; adding a volatile organic solvent to said first liquidhomogenate to form a two-phase mixture including an organic phase and asecond phase; thoroughly mixing the two-phase mixture for a timesufficient to permit substantially all of said extracted at least onetarget analyte to be extracted and collected into said organic phase;separating the organic phase from the second phase; and thereafter,reducing the volume of the separated organic phase to a fraction of itsoriginally separated volume by evaporating at least a portion of saidvolatile organic solvent and acid components therefrom to provide saidconcentrated extract.